Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.     

Actin-mediated retraction of the larval epidermis during metamorphosis of the sand dollar,Dendraster excentricus

Summary During the first 15 to 20 min of metamorphosis the larval arms are retracted and resorbed into the aboral surface of the juvenile. Arms excised from metamorphosing larvae will undergo a sequence of contraction and histolysis that is identical to that occurring in intact larvae. Prior to and during metamorphosis, epidermal cells contain bundles of 5 to 7-nm microfilaments in arrays radiating apically from the base of the cells. Sparse microfilaments also occur near the plasmalemma of epidermal cells and some mesenchymal cells in larvae fixed during metamorphosis. Contraction of excised arms is reversibly inhibited by treatment with cytochalasin B, and microfilaments bind myosin subfragment-l. Indirect immunofluorescence of larval arms using an antibody against chicken-muscle actin and staining with the F-actin specific probe, NDB phallacidin indicate that the arms contain actin distributed in a manner consistent with ultrastructural findings. It is proposed that retraction of the larval arms during metamorphosis is produced by an actin-mediated change in shape of the epidermal cells.     

Construction of a vector, pRSVcatamb38, for the rapid and sensitive assay of amber suppression in human and other mammalian cells.

We describe the generation of an amber mutation in the chloramphenicol acetyltransferase (cat) gene of the mammalian cell transfection vector pRSVcat (Gorman et.al. (1982), Proc.Natl.Acad.Sci. 79 6777-6791). We have demonstrated the in vivo suppression of this amber mutation in monkey and human cells by co-transfection with a synthetic Xenopus suppressor tRNATyr under the control of the late SV40 promoter. The vector, pRSVcatamb38, may be used to quantitate amber suppression in various mammalian cells.     

Inhibition of thyroidal cyclic AMP-dependent protein kinase by thyroid hormone.

Bovine thyroid cyclic AMP-dependent protein kinase was purified by DEAE-Sephadex and Sephadex G-200 chromatography. This preparation showed a 240-fold increase in specific activity over the initial 20,000 x g supernatant with histone as substrate and 1 micronM cyclic AMP in the assay mixture. In the presence of 2.5 X 10(-5)M L-triiodothyronine (T3), protein kinase activity was significantly reduced; 50% inhibition was achieved at 1 X 10(-4) M. Tests of diverse thyroid hormone analogs showed that T3 and its derivatives were more potent inhibitors than T4 and its derivatives which, in turn, were more potent than thyronine or diiodothyronine. Mono- and diiodotyrosine, tyrosine, and iodide were without effect. Triiodothyronine did not inhibit kidney, spleen, or lung protein kinase activity. The magnitude of the inhibition was the same whether or not cyclic AMP (1 micronM) was present in the incubation mixture, suggesting an effect on the catalytic, rather than the regulatory subunit of the enzyme. The inhibition of protein kinase by thyroid hormone was not influenced by Mg++ concentration but was overcome in a competitive manner by increasing ATP concentration. Increasing the histone concentration did not modify the inhibition. Although these studies suggest a novel cellular control mechanism, the high thyroid hormone concentrations required and the lack of concordance between inhibitory effects and biologic activity of the analogs tested precludes assumption of physiologic relevance.     

Sensitivity ofBacillus subtilis sporulation to methylene blue inhibition

The dye methylene blue was found to inhibit sporulation inBacillus subtilis 168. The compound blocked spore formation at concentrations subinhibitory to vegetative growth while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to the inhibitor through T₆, but germination and outgrowth were not affected by the presence of the compound. The inhibition of sporulation may be related to the ability of the compound to inhibit oxidative phosphorylation.     

CEA, ZGM and EMA localization in cells of pleural and peritoneal effusion: a preliminary study

Carcinoembryonic antigen (CEA), the zinc glycinate marker (ZGM) and epithelial membrane antigen (EMA) have been described as epithelial or tumour markers of varying specificity. These antigens were studied by immunoperoxidase localization in selected cell blocks of 62 pleural or peritoneal effusions and compared to cytological findings and review of the clinical records. By cytological criteria, 25 of the cell blocks were positive for malignancy, 30 negative, and 7 inconclusive. CEA, ZGM, and EMA by immunoperoxidase staining were localized on the cell surface and often in the cytoplasm of malignant cells, in 11/25 (44 per cent), 17/25 (68 per cent) and 22/25 (88 per cent) of the positive cell blocks respectively. Ten (40 per cent) of these cases were positive for all three antigens, 7 (28 per cent) for two, and 6 (24 per cent) for one. Of the 7 cases which were inconclusive on routine cytological reporting, 5 were positive for at least one marker. In 3 of the 5 a diagnosis of malignancy was confirmed, and in the other two was strongly suspected as malignant on clinical grounds. Macrophages were sometimes positive for one or more markers (but showed cytoplasmic staining only) and mesothelial cells in some cases stained positively for EMA but were always negative for CEA and ZGM. Localization of the 3 antigens in cells of malignant effusions was compared with their localization in the primary tumours in 9 cases. Localization corresponded for CEA in 7 of 9 cases, for EMA in 8 of 8 an for ZGM in only 2 of 9. Effusion fluid levels for CEA were compared with the cytological and immunocytochemical findings in 30 cases. Mucin stains performed on the cell blocks were also compared with the immunoperoxidase findings.     

Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies).

Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction.     

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.     

Measurement of superhelix densities in buoyant dye/CsCl. The use of a standard other than native SV40 DNA.

The dye-induced separation between closed and open duplex DNAs in buoyant CsCl is determined primarily by the superhelix density of the closed DNA, provided that all other experimental variables (such as the solution density and dye concentration) are held constant. The extent of the buoyant separation may be used to estimate the superhelix density of an uncharacterized closed DNA, by comparison with the corresponding separation with native SV40 DNAs under identical conditions. We present here an extension of these quantitative relationships to permit the use of an arbitrarily selected closed duplex DNA of known superhelix density, with the accompanying open form, as a reference. The general result is that the ratio of buoyant separations for any two closed/open DNA pairs remains a linear function of the difference in superhelix densities between the closed DNAs. The value of the proportionality constant depends, however, upon the magnitude of the superhelix density of the closed DNA selected as reference.