Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.     

Expression of A and B Types of Monoamine Oxidase in Differentiated Neuroblastoma Hybrid Cells

The total activities of monoamine oxidase (MAO) and the ratio of type B/type A activities were determined in mouse neuroblastoma N1E-115 cells, and in NX31T and NG108-15 hybrid cells derived from mouse neuroblastoma X rat sympathetic ganglion hybrid or mouse neuroblastoma X rat glioma hybrid cells. N1E-115 and NX31T cells possessed type A activities exclusively, although NG108-15 cells showed both type A (65-90%) and type B (10-35%) MAO activities. The activity of type A MAO in NX31T and N1E-115 cells was relatively constant during culturing periods in the presence or absence of dibutyryl cyclic AMP (Bt2cAMP), whereas total MAO activity and the ratio of type B MAO/type A MAO in NG108-15 cells increased as a function of culture periods. Prostaglandin E1 (PGE1) and theophylline, the best known combination to increase intracellular cyclic AMP content of NG108-15 cells, caused similar increases of MAO and of the type B/type A ratio in NG108-15 cells. The results suggest that MAO activity and expression of MAO B activity are regulated in NG108-15 cells in a cyclic AMP-dependent manner.     

Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

The concept of a changing receptor concentration: implications for the theory of drug action

Drugs are considered to produce their effects on biological tissues either by altering some physical property of cells or by interacting with specific cellular components, called receptors. Most drugs and endogenous neurotransmitters act on highly selective receptors located on the outer surface membrane of cells. These receptors were believed, until recently, to be stationary on the cell surface and to be present in unvarying numbers. Consequently, most early theorists modeled the drug-receptor interaction on the basis of stationary and static receptor molecules. The substantial advances in our understanding of drug action based on these models have partly justified this view. However, recent electron microscopic studies have revealed the presence of structures, including "coated" pits and vesicles, that appear to provide a mechanism by which cell surface receptors might be internalized in a process of endocytosis. The precise intracellular fate of these internalized receptors is unknown, but based on present understanding, it seems reasonable to believe that some are destroyed intracellularly whereas others are recycled to the cell surface. The importance of such processes to pharmacologic theory is a new awareness of a cellular pathway that is capable of internalizing drugs, receptors, or both. The implications of such a process to the theory of drug action extends to some unexplained drug phenomena such as down regulation, drug tolerance, tachyphyllaxis, and partial agonism. We present herein the theoretical framework for a model of drug action that incorporates the possibility of receptor internalization and subsequent degradation, recycling, or replacement.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Complexes prepared from protein A and human serum,IgG, or Fcγ fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion

Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of ¹²⁵I-protein A and human ¹³¹I-IgG alone or in serum, and ¹³¹1-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess ¹³¹I-IgG or ¹³¹1-Fc, all the ¹²⁵I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess ¹²⁵I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA protein A of Staphyloccus aureus - VBS EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4 - PBS 0.01 M phosphate buffered saline, pH 7.4     

The araBAD operon of Salmonella typhimurium LT2. I. Nucleotide sequence of araB and primary structure of its product, ribulokinase

Hybrid plasmids containing the araBAD operon of Salmonella typhimurium LT2 were characterized by Southern blot and genetic analyses. The nucleotide sequence of araB was determined. The araB gene product, ribulokinase (EC 2.7.1.16), was purified and the results of amino acid composition analysis and partial amino acid sequence are in agreement with predictions from the DNA sequence. Ribulokinase is 569 amino acid residues long and has a calculated Mr of 61 793. Ribulokinase shares significant homology with xylulose kinase from Escherichia coli. Codon usage in the araB gene does not favor those codons which have intermediate codon-anticodon binding energy.     

Open-field behaviour in chickens: A replication revisited

In an attempt to show that the open field can still be used as a valid measure of fear, Jones (1983) has reported a failure to replicate some of our findings. The present studies show that this was due to procedural and methodological differences. For instance, we found that birds tested in a novel environment behaved quite differently from those, as in Jones' case, which were placed in one resembling the home cage. Moreover, birds housed in isolation for two days prior to testing reacted differently than those, as again in Jones' case, which were reared in isolation from hatching to the time of testing. The results were interpreted as being consistent with our view that open-field behaviour reflects a conflict between the need to reinstate contact with conspecifics on the one hand, and evade predation on the other.