Overproduction of Escherichia coli replication proteins by the use of runaway-replication plasmids.

A derivative of the runaway-replication plasmid was constructed. This plasmid, pSY343, has the gene for kanamycin resistance and single sites for EcoRI, BamHI, HindIII, KpnI, and XhoI that can be used as cloning sites without inactivating the kanamycin resistance gene or the replication genes. Three replication genes of Escherichia coli were cloned on the plasmid. The activity of dnaA, dnaZ, and ssb gene products were 200-, 90-, and 60-fold greater, respectively, in the cells containing these plasmids than in normal cells.     

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

Congenitally defective aldosterone biosynthesis in humans: the involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients.

The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.     

Suppressive Effect of Liposomes Containing DNA Coding for Diphtheria Toxin A-Chain on Cells Transformed with Bovine Leukemia Virus

A recombinant plasmid which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) was constructed to test a novel application of liposomes as antiviral agents. The promoter activity of BLV-LTR was estimated by the chloramphenicol acetyltransferase (CAT) assay using a plasmid which contains the coding sequence of CAT under the control of BLV-LTR (pBLVCAT). When BLV-infected cells were transfected with pBLVCAT, CAT activity was detected. BLV-uninfected cell lines, however, showed no detectable CAT activity. The plasmid DNA entrapped in liposomes was added to BLV-infected cells in culture. Syncytium formation induced by BLV-infected cells was effectively suppressed by the liposomes containing the gene for DT-A under the control of BLV-LTR. Conversely, liposomes containing the gene for DT-A without a promoter showed no such effect. DT-A gene-containing liposomes with BLV-LTR did not affect formation of syncytium induced by bovine immunodeficiency virus. These observations indicate that BLV-infected cells were readily targeted on the level of gene expression. This strategy could be applied to the treatment of BLV-induced B-cell proliferation of cattle, and further to other viral/neoplastic diseases where specific gene expression is exerted.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.     

Protein kinase C in rat brain synaptosomes. Beta II-subspecies as a major isoform associated with membrane-skeleton elements.

A small fraction (approximately 5%) of protein kinase C (PKC) in the adult rat brain synaptosomes is tightly associated with Triton X-100-insoluble components (most likely membrane-skeleton elements), and is solubilized only after denaturation with sodium dodecyl sulfate. The kinase domain of this PKC can be released as a soluble form after limited proteolysis with calpain, whereas the regulatory domain which binds phorbol ester remains insoluble. The PKC in this fraction was identified as the beta II-subspecies or its related molecule. Presumably, this enzyme subspecies is responsible for the phosphorylation of a major PKC substrate protein, growth-associated protein-43, which is located in nerve endings as well as in growth cones in association with the membrane-skeleton elements.     

Secretory regulation of 19-hydroxyandrostenedione in normal man.

To evaluate the secretory regulation of 19-hydroxyandrostenedione (19-OH-AD), its plasma concentration was measured before and after stimulation and inhibition tests for the ACTH-adrenal axis and the renin-angiotensin system in 50 normal subjects. Basal levels of plasma 19-OH-AD did not correlate with either those of plasma renin activity (PRA) or the plasma aldosterone concentration (PAC), but positively correlated with those of plasma cortisol. Plasma 19-OH-AD was stimulated by 0.25 mg ACTH-(1-24) and was suppressed by 1 mg dexamethasone (DEX) as were plasma cortisol and PAC. On the other hand, with 2-h standing alone or iv 40 mg furosemide plus 2-h standing, plasma 19-OH-AD and cortisol did not increase but PRA and PAC did. With iv furosemide plus 2-h standing with 3 mg DEX pretreatment, plasma 19-OH-AD and cortisol did not respond either, but PRA and PAC increased. With 25 mg oral captopril following 1-h standing with 3 mg DEX pretreatment, plasma 19-OH-AD and cortisol did not change but PAC decreased. These results indicate that the secretion of 19-OH-AD is mainly under the control of the ACTH-adrenal axis rather than the renin-angiotensin system.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.