The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.     

Photosensory transduction in the flagellated alga, Euglena gracilis. III. Induction of Ca2+-dependent responses by monovalent cation ionophores

(1) The effects of monovalent cation ionophores (valinomycin and gramicidin), a protonophore (nigericin) and extracellular pH change on the motility and blue light-induced photobehavior (step-down photophobic response) of Euglena were investigated. (2) Monovalent cation ionophores, but not the protonophore, can both partially suppress photobehavior and, under appropriate conditions, induce a change in flagellar activity (and thus cell movement) that appears identical to that associated with the photobehavior. (3) Valinomycin, at low extracellular KCl, delays the induction of photobehavior and also induces a light-independent elevation in the frequency of directional changes in the cells' swimming path. Both effects are suppressed by elevation in extracellular KCl. (4) Gramicidin, in the presence of the anion tetraphenylborate, suppresses photobehavior. The same combination, if applied in the presence of elevated extracellular NaCl, induces a light-independent cell tumbling and elevation in the frequency of directional changes in the cells' swimming path. The induced behavior is dependent on the extracellular Na concentration, requires the presence of extracellular Ca²⁺ and is blocked by La³⁺. (5) Photobehavior is observed over the pH range 3.5–8.2 and fluence/response relationships for photobehavior are not significantly different over the pH range 5.5–8.2. (6) The results provide a link between the previously reported effects of Ca²⁺ ionophores, and the effects of monovalent cations and monovalent cation-transport inhibitor on motility and photobehavior.     

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M., Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194–6198), which in contrast to Ca²⁺ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca²⁺ and Na⁺ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na⁺ and Ca²⁺ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca²⁺ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca²⁺ efflux from sarcoplasmic reticulum vesicles which had been loaded with Ca²⁺ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H⁺, OH^?, or Cl^?.     

Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

Induction of histidine decarboxylase activity in mouse skin after application of indole alkaloids,a new class of tumor promoter

The effects of various promoters in two-step carcinogenesis on the induction of histidine decarboxylase in the skin of mice was investigated. The potencies of various phorbol esters in inducing histidine decarboxylase activity were parallel with their tumor-promoting activities. Indole alkaloids such as dihydroteleocidin B and lyngbyatoxin A, which induced ornithine decarboxylase and promoted tumor development in the skin of mice with the same potency as 12-O-tetradecanoylphorbol-13-acetate (TPA), also induced histidine decarboxylase activity. These results suggest that histamine produced by this inducible histidine decarboxylase may play some role in tumor promotion.     

人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因的修正及修正基因在大肠杆菌中的表达

本文采用寡核苷酸介导的定位诱变技术修正了人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因中出现的合成错误,在该基因编码区的201位插入了原来缺失的G残基,从而使之恢复正确读框。经对修正基因进行DNA全序列测定,表明定位诱变的结果符合设计要求。在此基础上,利用原核高效表达载体pBV220在P_RP_L串联启动子的控制下在大肠杆菌中对该基因进行了表达,并测定了重组人嗜中性白细胞活化蛋白-1/白细胞介素-8的嗜中性白细胞趋化活性。本工作为开展人嗜中性白细胞活化蛋白-1/白细胞介素-8基因工程及蛋白质工程的研究奠定了基础。     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.