An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase.

The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered.     

The dynamics of phase partition. A study of parameters affecting rat liver organelle partitioning in aqueous two-polymer phase systems.

Separation of subcellular organelles by two-phase partition is thought to reflect differential partition of the organelles between the two phases or between one of the phases and the interface. Studies by Fisher and colleagues [Fisher & Walter (1984) Biochim. Biophys. Acta 801, 106-110] suggest that cell separation by phase partition is a dynamic process in which the partition changes with time. This is mainly due to association of the cells with sedimenting droplets of one phase in the bulk of the other. Rat liver organelle partition was studied to determine whether the same dynamic behaviour is observed. Partition was clearly time-dependent during 24 h at unit gravity, and was also affected by altering the volume ratio of the two phases and the duration of phase mixing. These results indicate that, as with cells, the partition of organelles between phases is a dynamic process, and is consistent with the demonstration that organelles adhere to the phase droplet surfaces. Optimization of the volume ratio between phases may lead to significant processing economies. Organelle sedimentation in the upper phase was significantly faster than in the isoosmotic sucrose. Theoretical modelling of apparent organelle sizes indicates that aggregation occurs in the poly(ethylene glycol)-rich upper phase. This phenomenon is likely to limit the use of this technique in organelle separations unless means can be found to decrease aggregation.     

A new location for the human adenine phosphoribosyltransferase gene (APRT) distal to the haptoglobin (HP) and fra(16)(q23)(FRA16D) loci

The human adenine phosphoribosyltransferase gene (APRT) was mapped with respect to the haptoglobin gene (HP) and the fragile site at 16q23.2 (FRA16D). A subclone of APRT and a cDNA clone of HP were used for molecular hybridization to DNA from mouse-human hybrid cell lines containing specific chromosome 16 translocations. The APRT subclone was used for in situ hybridization to chromosomes expressing FRA16D. APRT was found to be distal to HP and FRA16D and was localized at 16q24, making the gene order cen-FRA16B-HP-FRA16D-APRT-qter.     

Methylammonium uptake by Rhodobacter capsulatus

The ammonium uptake system of Rhodobacter capsulatus B100 was examined using the ammonium analog methylammonium. This analog was not transported when cells were grown aerobically on ammonium. When cultured on glutamate as a nitrogen source, or when nitrogen-starved, cells would take up methylammonium. Therefore, in cells grown under nitrogen-limiting conditions, a second system of ammonium uptake (or a modified form of the first) is present which is distinguished by its capacity for transporting the analog in addition to ammonium. The methylammonium uptake system exhibited saturation kinetics with a K _m of 22 M and a V _max of about 3 nmol per min · mg protein. Ammonium completely inhibited analog transport with a K _i in the range of 1 M. Once inside the cell methylammonium was rapidly converted to -N-methylglutamine; however, a small concentration gradient of methylammonium could still be observed. Kinetic parameters reflect the effects of assimilation.The methylammonium uptake system was temperature and pH dependent, and inhibition studies indicated that energy was required for the system to be operative. A glutamine auxotroph (G29) lacking the structural gene for glutanime synthetase did not accumulate the analog, even when nitrogen starved. The Nif^- mutant J61, which is unable to express nitrogenase structural genes, also did not transport methylammonium, regardless of the nitrogen source for growth. However, the mutant exhibited wild-type ammonium uptake and glutamine synthetase activity. These data suggest that transport of ammonium is required for growth on limited nitrogen and is under the control of the Ntr system in R. capsulatus.Abbreviations CCCP carbonyl cyanide-m-chlorophenyl hydrazone - CHES cyclohexylaminoethanesulfonic acid - DMSO dimethyl sulfoxide - GMAD -N-methylglutamine - GS glutamine synthetase - MES 2-(N-morpholino) ethanesulfonic acid - MSX methionine-Dl-sulfoximine - pCMB p-chloromercuribenzoate - Tricine N-tris(hydroxymethyl)methylglycine     

An age dependent stochastic model of periodic screening: Basic properties

An age dependent stochastic model for the periodic screening of a progressive chronic disease is developed in this paper by combining results from previous modeling efforts. The basic concepts used are the random variables describing the disease free state and preclinical state sojourn times and the age at screening or observation, as well as generations of individuals defined according to time of entry into the preclinical state. At discrete time points, the model characterizes the density functions for individuals who are healthy, have preclinical disease, or have clinical disease. The joint density functions of age and sojourn times for cases detected by a periodic screening program and for cases which surface clinically between screens are derived as functions of screening interval, false negative rate, and disease natural history.     

Thermotropic lipid phase separations in human platelet and rat liver plasma membranes

Electron spin resonance (ESR) studies were conducted on human platelet plasma membranes using 5-nitroxide stearate, I(12,3). The polarity-corrected order parameter S and polarity-uncorrected order parameters S(T parallel) and S(T perpendicular) were independent of probe concentration at low I(12.3)/membrane protein ratios. At higher ratios, S and S(T perpendicular) decreased with increasing probe concentration while S(T parallel) remained unchanged. This is the result of enhanced radical interactions due to probe clustering. A lipid phase separation occurs in platelet membranes that segregates I(12,3) for temperatures less than 37 degrees C. As Arrhenius plots of platelet acid phosphatase activity exhibit a break at 35 to 36 degrees C, this enzyme activity may be influenced by the above phase separation. Similar experiments were performed on native [cholesterol/phospholipid ratio (C/P) = 0.71] and cholesterol-enriched [C/P = 0.85] rat liver plasma membranes. At 36 degrees C, cholesterol loading reduces I(12,3) flexibility and decreases the probe ratio at which radical interactions are apparent. The latter effects are attributed to the formation of cholesterol-rich lipid domains, and to the inability of I(12,3) to partition into these domains because of steric hinderance. Cholesterol enrichment increases both the high temperature onset of the phase separation occurring in liver membranes from 28 degrees to 37 degrees C and the percentage of probe-excluding, cholesterol-rich lipid domains at elevated temperatures. A model is discussed attributing the lipid phase separation in native liver plasma membranes to cholesterol-rich and -poor domains. As I(12,3) behaves similarly in cholesterol-enriched liver and human platelet plasma membranes, cholesterol-rich and -poor domains probably exist in both systems at physiologic temperatures.     

Decrease in mitogen responsiveness of mononuclear cells from peripheral blood after epinephrine administration in humans

A single subcutaneous injection of 0.2 mg epinephrine into healthy human subjects caused a transient lymphocytosis in peripheral blood. Mononuclear cells (MNC), isolated at various times after epinephrine administration, were cultured in the presence of mitogens. The blastogenic responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were significantly reduced for up to 60 min post-epinephrine (p less than 0.05); the response to concanavalin A (Con A) was reduced in the 15-min samples only. All responses returned to pre-injection levels by 120 min post-injection. Removal of adherent monocytes from MNC isolates before culture did not restore normal mitogen responsiveness. When MNC were cultured in the absence of mitogens, there was no difference in survival between pre- and post-epinephrine samples. Incubation of untreated MNC for 2 hr or 18 hr in vitro with various concentrations of epinephrine (10(-5) to 10(-1) mg/ml) had no effect upon the subsequent blastogenic response to mitogens. Other workers have reported that epinephrine administration causes alterations in the composition of the circulating lymphocyte pool. Taken together, these data suggest that the reduction in mitogen responsiveness after epinephrine is the result of changes in the distribution of lymphocyte subclasses in peripheral blood.     

Thymidylate synthetase inhibitors and fragile site expression in lymphocytes.

The ability of three thymidylate synthetase inhibitors, fluorodeoxyuridine, fluorodeoxycytidine, and trifluorothymidine, to induce the expression of eight different folate-sensitive fragile sites has been investigated in 22 patients and compared with the efficacy of simple folate deprivation for inducing fragile site expression. Fluorodeoxyuridine and fluorodeoxycytidine were equal in their ability to elicit fragile site expression but fluorodeoxycytidine proved less cytotoxic under comparable culture conditions. Both fluorodeoxyuridine and fluorodeoxycytidine were found to be more efficient than trifluorothymidine at comparable concentrations but less efficient than simple folate deprivation in eliciting fragile site expression in lymphocytes. Since the three inhibitors induced expression of eight different folate-sensitive fragile sites, it is likely that all folate-sensitive fragile sites have a common underlying mechanism of expression. The practical application of thymidylate synthetase inhibitors in the routine detection of heritable fragile sites is discussed.     

Heritable fragile sites on human chromosomes. X. New folate-sensitive fragile sites: 6p23, 9p21, 9q32, and 11q23

Four new folate-sensitive fragile sites are documented at 6p23, 9p21, 9q32, and 11q23. These have all been shown to be heritable except for the one at 9p21, which has been seen only in a single individual. As with the other autosomal fragile sites, these appear to be innocuous in heterozygotes.