Studies on tolerance induction in vitro. I. Production of immunologically tolerant rabbit spleen cells and the transfer of tolerance to untreated cells.

Tolerance was induced in rabbit spleen cells by incubation with solubilized T2 phage (S-T2)2 at 37degrees C. Spleen cells thus treated maintained normal responsiveness to an unrelated antigen, S-SP82. Transfer of tolerance was demonstrated in in vitro in that the addition of washed tolerant cells caused suppression of the response of untreated cells to an immunogenic dose of S-T2. Evidence is presented that this suppression is not due to the transfer of tolerogenic quantities of antigen. Spleen cell populations depleted of adherent cells were still capable of being made tolerant and of transferring tolerance.     

Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.     

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser?Pulses

Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.     

Ribosomal ribonucleic acids of cultured cells: A preliminary survey of differences among mammalian species detectable by polyacrylamide gel electrophoresis

Summary Ribosomal ribonucleic acids (rRNA's) of cultured cells from various species were compared by polyacrylamide gel electrophoresis. Electrophoretic mobility of the 28 S RNA component varied according to species. Human cell 28 S rRNA was distinguishable from that of monkey cells. The mobility of chimpanzee 28 S rRNA was identical to that from human cells, but different from that of monkey cells. Cat, goat, and swine cell 28 S rRNA's were distinguishable from one another and from both human and monkey cell rRNA's. The mobilities of bovine, rabbit, rat, hamster, and mouse cell 28 S rRNA's were virtually identical to that from monkey cells. No apparent correlation existed between the mobility of 28 S rRNA and the currently accepted phylogenetic position of the donors. Normal cells, tumor cells, and virus-infected cells from the same species did not differ, but in some instances embryonic and adult cells were distinguishable. The 18 S rRNA components did not show variations comparable to those of the 28 S rRNA components. This work was supported by the Office of Naval Research and the Bureau of Medicine and Surgery, United States Navy, under a contract between the Office of Naval Research and the Regents of the University of California.     

Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.     

Will fencing floodplain and riverine wetlands from feral pig damage conserve fish community values?

Installation of feral pig (Sus scrofa) exclusion fences to conserve and rehabilitate coastal floodplain habitat for fish production and water quality services remains untested. Twenty‐one floodplain and riverine wetlands in the Archer River catchment (north Queensland) were surveyed during postwet (June–August) and late‐dry season (November–December) in 2016, 2017, and 2018, using a fyke net soaked overnight (~14–15 hr) to test: (a) whether the fish assemblage are similar in wetlands with and without fences; and (b) whether specific environmental conditions influence fish composition between fenced and unfenced wetlands. A total of 6,353 fish representing twenty‐six species from 15 families were captured. There were no wetland differences in fish assemblages across seasons, years and for fenced and unfenced (PERMANOVA, Pseudo‐F < 0.589, p < .84). Interestingly, the late‐dry season fish were far smaller compared to postwet season fish: a strategy presumably in place to maximize rapid disposal following rain and floodplain connectivity. In each wetland, a calibrated Hydrolab was deployed (between 2 and4 days, with 20 min logging) in the epilimnion (0.2 m) and revealed distinct diel water quality cycling of temperature, dissolved oxygen and pH (conductivity represented freshwater wetlands), which was more obvious in the late‐dry season survey because of extreme summer conditions. Water quality varied among wetlands in terms of the daily amplitude and extent of daily photosynthesis recovery, which highlights the need to consider local conditions and that applying general assumptions around water quality conditions for these types of wetlands is problematic for managers. Though many fish access wetlands during wet season connection, the seasonal effect of reduced water level conditions seems more overimprovised when compared to whether fences are installed, as all wetlands supported few, juvenile, or no fish species because they had dried completely regardless of the presence of fences.