Non-invasive, kinetic measurements of [3H]nitrendipine binding to isolated rat myocytes by condensed phase radioluminescence

The binding of 3H-labelled drug molecules to membranes of living cells gives rise to photon emission from tryptophan residues at proteinaceous binding sites. This phenomenon, called condensed phase radioluminescence, has been used to measure non-invasively the kinetics of [3H]nitrendipine binding and dissociation on the same samples of cultured beating cardiac myocytes. Signal arose only from bound drug molecules. Binding was monoexponential (tau = 5.5 min) as was dissociation (14.3 min). Preincubating cells with non-radioactive nifedipine reduced the amplitude and rate of [3H]nitrendipine but not of [3H]dihydroalprenolol binding. The potential uses of this phenomenon are discussed.     

Electiveness of rapair of plaque-type mutations induced by hydroxylamine in phage kappa of serratia

Phage ? treated extracellularly with hydroxylamine (HA) was preadsorbed to hcr, exr or wild-type (HY) host cells and plated with HY indicator. 5 plaque mutation types were scored. The frequency distributions of the 5 mutation types (mutation spectra) differed with the hosts, the spectrum in exr especially deviating significantly from those in hcr and HY. This indicates electiveness of repair of certain (pre-) mutations within different genomic regions. HA treatment time (dose) influenced the spectra, too, owing to three mutation types giving linear (one-hit) dose curves and two giving parabolic (about two-hit) curves. The host type did not influence these curve shapes. These findings show that the number of HA hits depends on the genomic region where (pre-) mutations occur.Inactivation of phages as well as cells was strongest in exr compared with hcr and HY hosts (factor 1.3). In contrast, induction of all 5 mutation types was lower in exr (factor about 0.5) and hcr (factor about 0.8) than in HY. This indicates that both repair types (probably post- and pre-replicative) are needed for perfecting part of the HA-induced mutations. The part lacking in repair-defective hosts may be caused by lethality within these hosts of certain premutative lesions. The frequency of mixed compared with pure mutant clones was small. Its dose dependence may be due to recessive lethal lesions within the non-premutated DNA strand.     

Hydroxylamine-induced purple adenine (ad-3) mutants in Neurospora crassa. I. Characterization of mutants by genetic tests

The genetic effects of hydroxylamine (HA) on Neurospora crassa were studied in an effort to understand the difference between the results obtained on very simple prokaryotic systems and those obtained with mammalian systems. A 2-component heterokaryon was used to study the inactivation of conidia and the induction of recessive lethal mutations at specific loci and over the entire genome. The heterokaryon is heterozygous for 2 closely linked loci, ad-₃A and ad-₃B, in the ad-₃ region. Specific locus mutations can result from either point mutation or chromosome deletion. The results were as follows: (1) Both homokaryotic and heterokaryotic conidia had multi-hit survival curves, and there was no difference between the survival levels of the two as a function of treatment time. (2) The frequency of recessive lethal mutations in the ad-₃ region increased as the square of treatment time.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

Mutagenesis of yeast by hydrazine: dependence upon post-treatment cell division.

Hydrazine was found to be mutagenic for yeast (Saccharomyces cerevisiae) at exposures (concentration × time) ranging over nearly three orders of magnitude. Little or no forward mutation from CAN1 to can1 was detectable upon immediate plating following treatment in neutral buffer suspension. Post-treatment cell division in yeast extract peptone dextrose complex growth medium was required for expression of induced mutation to canavanine resistance. Frequencies of induced mutation rose to levels approximately 10-fold higher than spontaneous levels for exposures between 0.1 and 12.0 min mol/l. Survival remained at 100%. For exposures greater than 80 min mol/l viability and mutation frequency began to decrease sharply. By contrast, single treatments of ethyl methanesulfonate, methyl methanesulfonate, N-methyl-N′-nitro-N-nitro-soguanidine, nitrous acid, hydroxylamine, and ultraviolet light were able to increase mutation frequency with this system upon immediate assay. Further growth-dependent increases in mutation frequency were not observed with HA and UV.Expression of HZ-induced mutation was detectable after treated cells had undergone less than one population doubling in YEPD. Such mutation expression could be blocked by the inhibitors cycloheximide and hydroxyurea, which block protein synthesis and DNA synthesis respectively. Results were similar to those obtained previously with Haemophilus influenzae and similarly suggest that, in this eukaryote, HZ-induced lesions lead to mutation by causing base mispairing at DNA replication rather than by means of an error-prone repair mechanism.     

Investigations on the electron transfer processes in Photosystem II: New insights by chlorophyll fluorescence decay measurements with additional saturating light pulses

Electron transfer processes in leaves were investigated by chlorophyll fluorescence decay measurements. A fast chlorophyll fluorescence decay was observed in the intact state, reflecting normal electron transfer in Photosystem II. After treatment with DCMU a slow chlorophyll fluorescence decay was measured due to blocked electron transfer after the primary quinone Q_A. Additional saturating light pulses, one between each two measuring pulses, were used to completely reduce Q_A of the intact leaf: the chlorophyll fluorescence decay became similar to that of a DCMU treated leaf. A decreased electron donation rate to the reaction centre P680 was obtained after treatment with hydroxylamine. The intensity of the additional saturating light pulses was not sufficient to reduce all Q_A under this condition and only a small increase of the average chlorophyll fluorescence decay time occurred. Following our previous paper [Berg et al. (1997) Photosynthetica 34, in press], we investigated the effects of water stress with the additional saturating light pulses. An almost complete reduction of Q_A was possible after water stress started. A small, but systematic shortening of the slow chlorophyll fluorescence decay followed, up to a relative loss of leaf mass of 80%. At this time a rapid shortening of the chlorophyll fluorescence decay occurred, caused by an electron deficiency at the donor site of PS II. Additional saturating light pulses had no effects on the chlorophyll fluorescence decay any more, revealing a radiationless recombination between the reduced primary quinone Q and the oxidized reaction centre P680⁺.     

Formation of Free Radicals and Protein Mixed Disulfides in Rat Red Cells Exposed to Dapsone Hydroxylamine

The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS–NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS–NOH in the presence of the spin trap, 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS–NOH (25–50 μM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO–OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS–NOH-generated DMPO–OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS–NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS–NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia. © 1997 Elsevier Science Inc.     

Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase

Pseudomonas strain PB16, a Gram-negative heterotrophic nitrifying bacterium closely related to Pseudomonas azalaica on the basis of 16 S rDNA analysis, was able to use hydroxylamine as an additional energy source during growth in acetate limited chemostat cultures giving an increased biomass yield. In aerobically growing cells of Pseudomonas PB16 only 50% of supplemented hydroxylamine could be recovered as nitrite. In addition to nitrite, N2O could be detected in the chemostat off-gas, indicating combined heterotrophic nitrification and aerobic denitrification. The maximum specific hydroxylamine oxidizing activity observed was 450 nmol per min per mg dry weight, with a Ks of approximately 40 µm. Upon addition of hydroxylamine to the medium, Pseudomonas PB16 induced a soluble 132 KDa dimeric hydroxylamine oxidoreductase. The enzyme had a pH optimum of 9, and did not contain spectroscopic features typical for cytochromes, which is in contrast to hydroxylamine oxidoreductases fou nd in autotrophic bacteria.     

Reactions Between Dipeptidyl Peptidase Iv and Diacyl Hydroxylamines: Mechanistic Investigations

Abstract

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of K_cat/K_Inact

Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship.

Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO₂) and the decomposition of the suicide substrate in H₂O and D₂O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.