Hélène Charniaux-Cotton (1918–1986)

Summary

1-Methyladenine (1-MA) secreted from the follicle cells is the biological signal for meiosis reinitiation of starfish oocytes. The signal of-1-MA is transduced into cytoplasmic formation of maturation-promoting factor (MPF) that eventually induces a germinal vesicle breakdown (GVBD). Microinjection of pertussis toxin (PTX) inhibited 1-MA-induced GVBD in Asterina pectinifera and Asterina (Patina) miniata. PTX-inhibition of GVBD was rescued by the injection of MPF into PTX-preinjected oocytes. Most of the PTX- and MPF-double injected eggs were fertilized and underwent cleavage, suggesting the presence of a GTP-binding protein (G protein) specific for 1-MA signal transduction. Indeed, plasma membrane preparations of A. pectinifera oocytes contained a G protein consisting of 39-kDa α, 37-kDa β, and 8-kDa γ subunits. The α subunit contained a site for ADP-ribosylation catalyzed by PTX. It was also recognized by antibodies specific for a common GTP-binding site of mammalian α subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G protein (G_i) α subunits. Its gene was 74% and 83.7% identical to the rat G_i-2α gene in nucleotide and deduced amino acid sequences, respectively. The 39-kDa α subunit shared the common GTP-binding site of mammalian G protein α subunits and the PTX-catalyzed ADP-ribosylation site of mammalian G_i α subunits as expected from the immunoreactivity. The oocyte membranes had apparently two forms of 1-MA receptors with high and low affinities. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. The 39-kDa α subunit of starfish G protein was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. These results indicate that in starfish oocyte membranes, 1-MA receptors are functionally coupled with the 39-kDa PTX-substrate G protein that transduces the signal into the formation of a cytoplasmic factor (MPF) and eventually into the reinitiation of meiosis.     

Microbial Transformation of Sterols

The linear double stranded DNA plasmid pGKLl encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood. We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex. These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups. The iki2 mutation was mapped to a position near the centromere on chromosome XIII. We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Sccharomyces gene library, and isolated three DNA fragments that complement the ikil, iki3, and iki4 mutations, respectively.     

Mutation induction by okadaic acid, a protein phosphatase inhibitor, in CHL cells, but not in S. typhimurium.

Okadaic acid (OA) is a specific and strong inhibitor of protein phosphatase 1 and 2A present in eukaryotes, and a potent promoter of carcinogenesis in mouse skin. In this study, we examined the mutagenicity of OA. OA did not induce mutations in S. typhimurium TA100 and TA98, with or without a microsomal metabolic activation system. However, it was strongly mutagenic to Chinese hamster lung (CHL) cells without a microsomal activation system, as shown using diphtheria toxin (DT) resistance (DT^r) as a selective marker. Treatment of CHL cells with OA at 17.5 ng/ml induced 164 DT^r mutants per 10⁶ survivors. A plot of the mutation frequency against the OA concentration gave a concave curve, and the mutant frequency was calculated to be 5500/10⁶ survivors/μg, with OA in the dose range of 10–15 ng/ml. This value was about 680 times that of ethyl methanesulfonate (EMS), and comparable to that of 2-amino-N⁶-hydroxyadenine, one of the strongest knowon mutgens. Elongation factor 2 (EF-2) obtained from 4 DT^r clones was not ADP-ribosylated by DT fragment A. PCR-direct sequencing revealed that the hot spot of EF-2 for EMS mutagenesis in CHO-K1 cells, the first letter of codon 717, was not a t spot for OA mutagenesis in CHL cells.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets

In rat pancreatic islet membranes exposed to [alpha-32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively. In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit.     

一种由抗TF抗原的McAb和Daunomycin构建的免疫霉素在体外对人癌细胞的杀伤作用

在纯化了的抗TF(Thomsen—Friedenreich)抗原的McAb49H.8和抗肿瘤药物道诺霉素(Daunomyciu,DM)之间通过酸性敏感的顺式乌头酰基(Cis—Aconityl,CA)配接成免疫毒素49H.8—CA—DM.通过DNA合成抑制检测了此配接物在3个人癌细胞株(647V,EJ—S—47, LOVO)和一种小鼠肺癌细胞株KLN205的体外抗肿瘤效应.此免疫毒素显示了有相对选择性的细胞杀伤作用,对49H.8抗原强阳性的胱膀癌细胞株647V有确定的细胞杀伤效应,且呈剂量依赖性的,而在同一剂量范围对细胞表面缺乏49H.8抗原的小鼠肺癌细胞株KLH205不显示细胞毒性作用,对49H.8抗原弱阳性的细胞株EJ—S—47和LOVO的作用是不恒定的.     

Complementation of a DnaK-deficient Escherichia coli strain with the dnaK / dnaJ operon of Brucella ovis reduces the rate of initial intracellular killing within the monocytic cell line U937

Abstract To determine the degree of heterogeneity among Shiga-like toxin-II (SLT-II)-related toxins present in enterohemorrhagic Escherichia coli O157 strains, slt-IIB -related genes of 15 strains were amplified and sequenced. Of these 15 isolates, six contained only the slt-II -related genes, seven strains harbored slt-II -related genes together with slt-II , and two strains had slt-II -related genes plus slt-I . In strains carruing slt-II -related genes alone or in combination with slt-I , the PCR fragments were directly subjected to Taq cycle sequence analysis. Direct sequencing was not possible with the seven strains possessing both slt-II and slt-II -related genes, since the PCR products contained both genes. In order to allow sequence analysis of these slt-II -related genes, the PCR products were first subjected to restriction enzyme digestion with Fok I, which selectively digested slt-IIB . This resulted in an undigested 270-bp fragment consisting of pure slt-II -related genes. Interestingly, comparison of the nucleotide sequences revealed 100% homology of all analyzed 15 slt-IIB -related toxin genes. In addition, the nucleotide sequence of slt-IIB -related toxin genes were identical to slt-IIcB . Our findings indicate that SLT-IIc is a major variant form of SLT-II present in E. coli O517 strains.     

Rapid Communication: The Role of P-Type Calcium Channels in the Depolarization-Induced Activation of Nitric Oxide Synthase in Frontal Cortex

Abstract: In this study we demonstrate that 50 mRS K⁺ stimulates the conversion of L-[³H] arginine to L-[³H] citrulline and that this effect is blocked by 10 μ M AT-nitro- l -arginine, a nitric oxide synthase inhibitor, and Ca²⁺-free conditions. Amiloride (1 m M ) and low Na⁺ conditions were used to test the possible involvement of the Na⁺-Ca²⁺ exchanger. These treatments were without effect. The calcium channel blockers 10 mRS Mg²⁺, 100 μ M Cd²⁺, and 10 mRS Co²⁺ also blocked the K⁺ response, suggesting the involvement of voltage-dependent calcium channels (VDCCs). The specific VDCC involved seems to be the P type, as funnel-web spider toxin blocked the response whereas 200 μ M Ni²⁺, 10 μ M nifedipine, and 100 n M ω-conotoxin did not.     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.