Corticosteroidogenesis in the hypertrophic adrenal gland produced by betamethasone 17, 21-dipropionate in the rat fetus

Betamethasone 17, 21-dipropionate (BMDP), a potent glucocorticoid, produces adrenal hypertrophy in the rat fetus. The present study was performed to investigate the possible alterations of corticosteroidogenesis due to endogeneous substrates or exogenous pregnenolone in the incubation of homogenates of fetal hypertrophic adrenals caused by BMDP given to pregnant rats at day 19 of pregnancy.The corticosteroidal products and those levels per mg homogenate in an incubate of the hypertrophic adrenal homogenate did not differ from those of a normal adrenal. No accumulations of abnormal precursors or intermediates were found in the incubates of the hypertrophic adrenals. It is concluded from these findings that no qualitative alterations in the pathway of corticosteroidogenesis occurred in the hypertrophic adrenal glands caused by BMDP in the rat fetus. When the calculation was done per adrenal gland, the content of corticosterone in the incubate of the homogenate of the hypertrophic adrenal was remarkably higher than that found in a normal gland. This finding was compatible with the significant increase of the plasma corticosterone concentration in the fetuses with the adrenal hypertrophy caused by BMDP.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.     

Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.     

Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coli. Relation to the orientation of membrane proteins

The introduction of positive charges at the amino terminus of the mature domain of secretory proteins resulted in strong inhibition of their translocation across the cytoplasmic membrane of Escherichia coli, both in vitro and in vivo. The model secretory proteins used were OmpF-Lpp chimeric proteins possessing a cleavable or uncleavable signal peptide, beta-lactamase (Bla) and Bla-Lpp chimeric proteins. It is suggested that positively charged residues preceding the hydrophobic domain of the signal peptide have a positive effect, and ones following the hydrophobic domain, a negative effect on the translocation. These findings are discussed in relation to the orientation of membrane proteins, of which positive charges are predominant on the cytoplasmic surface.     

Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli

Expression of the ompF and ompC genes, which encode the major outer membrane proteins, OmpF and OmpC, respectively, is affected in a reciprocal manner by the osmolarity of the growth medium. This osmoregulation is mediated by the OmpR protein, a positive regulator of both genes, which is encoded by the ompR gene. Structural and functional properties of this regulatory protein were studied through complementation analysis of the wild-type and five mutant ompR genes that exhibited differences in osmoregulation of the expression of the OmpF and OmpC proteins. Complementation was carried out with combinations of a host strain and a plasmid, each of which carried either the wild-type or a mutant ompR gene. In some combinations, negative complementation was observed. For example, ompR1, a deletion mutation with an OmpF- OmpC- phenotype, was dominant to OmpF+ or OmpC+ phenotypes conferred by other ompR genes. Positive complementation of two mutant ompR genes was also observed in other combinations, when the two mutations were distantly located from each other on the OmpR protein. These results, together with other observations, support the view that the OmpR protein has a two-domain structure, each domain exhibiting a different role in the expression of the OmpF and OmpC proteins, and that this protein takes a multimeric structure as a functional unit.     

Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli

A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed. Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala-Glu residue. The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan. These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side. The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents. The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein.     

Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.