The interactions between nucleic acids and polyamines. III. Microscopic protonation constants of spermidine

Changes in the NMR chemical shifts of protons adjacent to the nitrogen atoms of Spermidine which are undergoing protonation have been measured by two-dimensional heteronuclear coupled NMR. Data thus obtained measure the dependence of the state of protonation of individual nitrogens on the pH, and permit calculation of the microprotonation constants of Spermidine and the concentrations of all of the variously protonated Spermidine species present at any pH.     

A technique for integrating any DNA fragment into the chromosome of Escherichia coli

We describe a technique that allows the insertion of any DNA fragment into the EcoRI-site-containing malPpa, the promoter of malPQ, one of the three maltose operons of Escherichia coli. DNA fragments were cloned into the unique EcoRI site of the pBR322-derived plasmid pOM40, which carries malPpa. In the next step these fragments were transposed into the chromosome by homologous recombination events occurring on both sides of malPp. Cells in which such insertion of the entire recombinant plasmid have occurred can be conveniently selected. Excision and curing of the vector plasmid could then occur spontaneously at a high frequency, leaving behind the inserted fragment that can be manipulated as any chromosomal marker. When the inserted fragment contains a properly positioned promoter, its promoting activity can be estimated by assaying amylomaltase, the product of malQ. When required, the inserted fragment can be easily transferred back onto pOM40. As examples of application we have transferred two different fragments into the chromosome of E. coli: one contained the ceaC-ceiC operon, which encodes colicin E3 and its immunity protein, and the other contained the lac promoter of E. coli.     

Effects of chronic hyperinsulinaemia on hepatic enzymes involved in lipogenesis and carbohydrate metabolism in the young rat.

Chronic (6 days) hyperinsulinaemia in young rats produced lower blood glucose concentrations and augmented body- and liver-weight gain. The insulin-treated rats had increased hepatic activities of citrate-cleavage enzyme, 'malic' enzyme and high-substrate (6.6 mM-phosphoenolpyruvate) pyruvate kinase, and decreased glucose 6-phosphatase. There were no changes in activities of phosphoenolpyruvate carboxykinase, phosphofructokinase, low-substrate (1.3 mM-phosphoenolpyruvate) pyruvate kinase, glucokinase and hexokinase.     

In vivo binding of 3H-pimozide in mouse striatum: Effects of dopamine agonists and antagonists

Several lines of evidence indicate that the i.v. injection of ³H-pimozide results in a specific in vivo binding of the neuroleptic to dopaminergic receptors.First, ³H-pimozide is preferentially accumulated in the striatum as compared to non-dopaminergic structures like the cerebellum. Second, the selective accumulation of ³H-pimozide is prevented by prior administration of various neuroleptics as well as by apomorphine. Moreover, doses of antagonists which prevent this accumulation were identical to those which lead to an increased striatal HVA level. Third, ³H-pimozide accumulation is not modified by the administration of a variety of non-dopaminergic agents.However, ³H-pimozide binding is not prevented either by indirect dopamine agonists and is even greatly increased by d-amphetamine at high doses. The possibility that direct or indirect dopamine agonists may favour the binding of the antagonist through a modification of receptor sites is discussed.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Microbial production of 4,4'-dihydroxybiphenyl: biphenyl hydroxylation by fungi

Of 15 species of fungi examined for their ability to hydroxylate biphenyl, 10 produced 4-hydroxybiphenyl. Seven of the 10 also produced 4,4'-dihydroxybiphenyl. The most efficient strains, Absidia pseudocylindrospora NRRL 2770 and Absidia sp. NRRL 1341, were more closely examined to determine their growth characteristics and the kinetics of biphenyl hydroxylation in batch fermentation. In the absence of biphenyl, A. pseudocylindrospora 2770 and Absidia sp. 1341 grew about as rapidly and efficiently in a defined glucose minimal medium as in a complex medium. Substrate yield coefficients for glucose in both media were 0.4 to 0.5 g of biomass per g of glucose, and the specific growth rate was about 0.17 h (doubling time, about 4 h). In this unoptimized system, 10 to 15 g of biomass per liter (dry weight) could be produced, using a defined salt solution and glucose as sole carbon and energy source. In the presence of biphenyl, growth was inhibited, more so for strain 1341 than for strain 2770. However, the specific activity for biphenyl hydroxylation (milligrams of biphenol per gram of biomass) was about 3.5 times greater for strain 1341. Furthermore, biphenyl hydroxylation appeared to require the presence of an oxidizable carbon and energy source (and perhaps growth) to proceed and, at least for strain 1341, hydroxylation seemed to be more efficient in the complex medium.     

Permeability characteristics of muscle membrane

Unidirectional flux rates of saturated fatty acids, saturated alcohols, and bile acids were measured in an intact rat diaphragm preparation. The logarithm of the permeability coefficients for fatty acids containing from five to ten carbon atoms was a linear function of the number of carbon atoms in the fatty acid chain. Incremental free energies of solution were +336 cal · mol⁻¹ for the addition of a hydroxyl group and −258 cal · mol⁻¹ for the addition of a methylene group. These incremental free energies were similar to those obtained by other investigators in other animal tissues, and our data suggest a structural similarity between membranes in different tissues and in different species. The muscle membrane exhibited anomalously high permeabilities for fatty acids containing less than five carbon atoms. Since muscle lacks tight junctions, this result suggests that small non-electrolytes traverse polar regions or aqueous pores within the cellular membrane.