Crystal structure of human DT-diaphorase: a model for interaction with the cytotoxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954).

The crystal structure of human DT-diaphorase (NAD(P)H oxidoreductase (quinone); EC 1.6.99.2) has been determined to 2.3 A resolution. There are only minor differences in shape and volume between the active sites of the rat and human enzymes and in the hydrophobic environment in the vicinity of the substrate. The isoalloxazine ring of the bound FAD is more buried in the human structure. Molecular modeling was used to examine optimal positions for the antitumor prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) in both the human and rat enzyme active sites. This suggests that the position of CB1954 in the active site of the human enzyme is very similar to that in the rat, although there are detailed differences in the predicted patterns of hydrogen bonding between side chains and the drug. Some of the differences are a consequence of the shift in position for the FAD molecule and may contribute to the observed differences in rate of the two-electron reduction of CB1954.

Oat bran, barley and malted barley lower plasma cholesterol relative to wheat bran but differ in their effects on liver cholesterol in rats fed diets with and without cholesterol.

Males rats were fed diets containing wheat bran, oat bran, barley or malted barley without or with 10 g/kg cholesterol + 1 g/kg cholic acid (referred to as "cholesterol"). Plasma total, HDL, and VLDL+LDL cholesterol concentrations were higher overall in rats fed cholesterol. There was a significant interaction between dietary cholesterol and cereal type. Higher concentrations of total and VLDL+LDL cholesterol were found in rats fed cholesterol with wheat bran than in those fed oat bran, barley or malted barley. HDL cholesterol concentrations were higher overall in rats fed wheat bran than in those fed oat bran or barley but not malted barley. Liver cholesterol pools were higher overall in rats fed cholesterol. In all animals fed oat bran, liver cholesterol was lower than in rats fed barley or malted barley. Hepatic HDL receptor activity was lower overall in rats fed cholesterol. There was no independent effect of cereal type on HDL receptor activity, but there was a significant interaction with dietary cholesterol. Activity tended to be higher in rats fed malted barley with cholesterol than in rats fed malted barley without cholesterol. LDL receptor activity was not affected by cereal type but was significantly lower overall in rats fed cholesterol.

Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy.

The intrinsic fluorescence properties of the oncogene protein p21N-ras,p21H-ras and one of its transforming mutants, p21N-ras (Val12), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect.

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

A comparison of heat-induced gel strengths of bovine plasma and egg albumen proteins.

The heat-induced gel strength in suspensions of two types of plasma protein isolates and egg albumen were compared to investigate the use of bovine blood plasma in food systems as a replacement for egg albumen and other proteins. A viscosity index, based on a counter-flow back-extrusion model, was used to measure gel strength. The optimum pH for gel formation was 7.0 for phosphated and nonphosphated plasma protein suspensions and 9.0 for egg albumen. The protein gel strengths were compared at 8% protein concentrations and at their respective pH optima. The gel strength of heated bovine plasma protein suspensions was greater (P < .01) than that of egg albumen, indicating that blood plasma exhibited a binding ability superior to that of egg albumen. The addition of controlled low levels of sodium and calcium increased (P < .05) the binding ability of both plasma protein isolates, while egg albumen showed no changes (P > .05). Greater concentrations substantially decreased (P < .01) the gel strength in both types of heated plasma protein suspensions and egg albumen, revealing that all three protein types exhibited an ionic strength dependency.

Evaluation of ten potential organic spermicides.

The following compounds, selected as possible vaginal or cervical spermicides, were tested in vitro for their ability to decrease the motility of human spermatozoa: cytochalasins A and B, N-cetylpyridinium chloride (CPC), N-cetyl-N,N,N-trimethylammonium bromide (CTAB), sodium deoxycholate (SDC), quinine and emetine hydrochlorides, 4-nitrophenyl-4-guanidinobenzoate hydrochloride, n-hexanal, and 6-amidino-2-(4-amidinophenyl) dilactate. The most effective spermicide was the antibacterial cytochalasin A which instantaneously abolished motility at a concentration of 0.01%. The two cationic detergents CPC and CTAB were also very potent and instantly immobilised spermatozoa at a concentration of 0.1%. Quinine, emetine and 4-nitrophenyl-4-guanidinobenzoate hydrochlorides at a concentration of 0.1% abolished motility in 30-60 minutes. n-Hexanal, SDC, and the antihaemostatic compound 6-amidino-2-(4-amidinophenyl) dilactate at 0.1% depressed motility but never rendered the sperm completely immotile. Cytochalasin B had little or no effect on sperm motility even at 0.1%.

PIP: In vitro studies were conducted with 10 potential organic spermicides to test their ability to decrease sperm motility. The organic substances tested were: cytochalasins A and B, N-cetylpyridinium chloride (CPC), N-cetyl-N,N,N-trimethylammonium bromide (CTAB), sodium deoxycholate (SDC), quinine and emetine hydrochlorides, 4-nitrophenyl-4-guanidinobenzoate hydrochloride, n-hexanal, and 6-amidino-2-(4-amidinophenyl) dilactate. The 1st 2 substances are antibiotics secreted by at least 4 different orders of fungi; the next 3 substances are soaps or detergents; quinine derives from the bark of the cinchoa tree and emetine derives from ipecac. The most effective spermicide was the antibacterial cytochalasin A which severely reduced the percentage of motile sperm even at quite low concentrations and caused an instantaneous loss of motility at higher concentrations. The rapidity of the effect and the low concentrations required make this agent a potentially useful spermicide. 2 of the detergent agents, CPC and CTAB, were also very potent, instantly immobilizing sperm at a .1% concentration. All the other agents tested had some spermicidal effect except cytochalasin B which had no effect on sperm motility even at .1% concentration.

The concentrations of free L-carnitine and L-O-acetylcarnitine in spermatozoa and seminal plasma of normal, fresh, and frozen human semen.

Free-L-carnitine and L-O-acetylcarnitine concentrations have been determined in spermatozoa and seminal plasma of normal, fresh, and frozen human semen. Results show that in fresh semen most of the free L-carnitine (0.213 +/- 0.02 mM) and L-O-acetylcarnitine (0.063 +/- 0.007 mM) is found in the seminal plasma. In contrast to other worker's results, the concentrations of free L-carnitine and L-O-acetylcarnitine in spermatozoa were found to be high and were 0.384 +/- 0.066 mumole/10(9) spermatozoa (22 mM) and 0.376 +/- 0.057 mumole/10(9) spermatozoa (21.6 mM), respectively. It is also demonstrated that the distribution of soluble metabolites such as L-carnitine between spermatozoa and seminal plasma is altered by the freezing of semen. After freezing and storage of semen at -20 degrees C for 7 days, the intracellular concentrations of free L-carnitine and L-O-acetylcarnitine decreased to below the limits of assay.