The regiochemistry and stereochemistry of the biosynthesis of vitamin B6 from triose units

13C and 2H NMR spectroscopy has been employed to probe the biosynthesis of vitamin B6 in Escherichia coli. The 13C NMR spectrum of a sample of pyridoxol derived biosynthetically from D-[1,2,3,4,5,6-13C6]glucose shows that the bonds, C(2)-C(3) and C(4)-C(5), of the pyridine nucleus are the only two carbon-carbon bonds of pyridoxol which are generated de novo in the course of its biosynthesis from glucose. It follows that the pyridoxol skeleton is generated from two intact triose units and a triose-derived two-carbon unit, all of which are supplied by glucose. From the 2H NMR spectra of samples of pyridoxol derived from (R)-[1,1-2H2]glycerol and (S)-[1,1-2H2]glycerol, respectively, it can be deduced that the rehydroxymethyl group of glycerol enters C-2', C-4', and C-5' of the pyridoxol skeleton. It follows that each of the three fragments is derived from glycerol in stereo-specific fashion. These results answer questions concerning the regiochemistry and the stereochemistry of pyridoxol biosynthesis.     

Stereoselective formation of benzo(c)phenanthrene (+)-(3S,4R) and (+)-(5S,6R)-oxides by cytochrome P450c in a highly purified and reconstituted system

The principal oxidative metabolites formed from benzo(c)phenanthrene (B(c)Ph) by the cytochromes P450 in liver microsomes from control and treated rats are the 3,4- and 5,6-arene oxides. A procedure is described which allows determination of the enantiomer composition and absolute configuration of these arene oxides based on HPLC separation of isomeric thiolate adducts formed with N-acetyl-L-cysteine in base. Incubation of [3H]-B(c)Ph with highly purified cytochrome P450c in a reconstituted monooxygenase system followed by trapping of the metabolically formed arene oxides as above indicated that the 3,4-oxide was predominantly the (+)-(3S,4R)-enantiomer (90%) and that the 5,6-oxide consisted mainly of the (+)-(5S,6R)-enantiomer (76%). The results are discussed in terms of their implications about the catalytic binding site of cytochrome P450c.     

HPLC-purified 2-[125I]iodomelatonin labels multiple binding sites in hamster brain

Binding of 2-[125I]iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Production of Alkaline Phosphatase from Escherichia coli in Continuous Culture

A 2-liter continuous culture for the production of Escherichia coli rich in alkaline phosphatase is described. The maximal output is greater than 250 mg of enzyme per day. The enzyme yield per unit of bacterial dry weight is only one-fifth of that obtained in earlier investigations. However, because of the increased bacterial density, the output per unit volume of culture is more than four times as great.     

Embryonic and larval development of brown trout, Salmo trutta L.: exposure to trace metal mixtures in soft water

Freshly fertilized ova of brown trout, Salmo trutta L., were exposed to all possible mixtures of Al (6000 nmol 1¹), Cu (80 nmol 1⁻¹), Pb (50 nmol 1⁻¹) and Zn (300 nmol 1 ¹). In a separate experiment, newly hatched brown trout yolk-sac fry were exposed to Mn (1500 nmol 1⁻¹), Fe (2500 nmol 1 ¹), Ni (200 nmol 1⁻¹) or Cd (4 nmol 1 ¹), separately, and in mixtures with either Al or Cu. Both experiments were conducted in flowing, artificial softwater media nominally at pH 5.6 [Ca] 20 μmol 1 ¹ and 10° C.
Mortalities were high in fry subjected to treatments which contained both Al and Cu (31–72%), and to the Cu + Fe treatment (78%) compared with those from the other trace metal mixtures (0–22%). In all the treatments tested, fry exposed to trace metal mixtures containing Al and/or Cu had reduced whole body Ca, Na and K content, and seriously impaired skeletal calcification. Whole body Mg content was variable. In trace metal mixtures which contained Cu but not Al, the effects on fry survival and whole body mineral content were in general more deleterious than the corresponding mixtures but with Al present rather than Cu. The presence of Pb and/or Zn in mixtures with Al and/or Cu had a slight ameliorative effect in terms both of fry survival and whole body mineral content.     

Episodic exposure to acid and aluminium in soft water: survival and recovery of brown trout,Salmo trutta L.

Yolk-sac fry, swim-up fry and 1–2 yr juveniles of brown trout, Sulmo trutta L., were exposed to episodes of aluminium and low pH, maximum aluminium concentration 12 μmol l⁻¹ (323 μg l⁻¹), minimum pH 4.5, total duration up to 54 h (yolk-sac fry) or up to 78 h (swim-up fry and juveniles), in an artificial soft water medium, [Ca] 20 μmol l⁻¹ (0–8 mg l⁻¹) (nominal baseline: pH 5.6, zero aluminium concentration). Yolk-sac fry mortality was nil or very low. A marked increase in susceptibility, with high mortalities, occurred when the yolk was fully absorbed. Mortality of juveniles exposed to two successive episodes was lower than would have been expected on the basis of comparisons with mortalities in single episodes, and mortality declined as the interval between the two episodes was increased. Disturbance of sodium, potassium or calcium balance or gill damage in surviving yolk-sac fry or juveniles was still evident 5 to 6 days after the end of a single episode.     

Growth in utero and cognitive function in adult life: follow up study of people born between 1920 and 1943.

OBJECTIVES--To examine the relation between fetal growth and cognitive function in adult life. DESIGN--A follow up study of men and women whose birth weights and other measurements of body size had been recorded at birth. SETTING--Hertfordshire, Preston, and Sheffield. SUBJECTS--1576 men and women born in Hertfordshire, Sheffield, or Preston between 1920 and 1943. MAIN OUTCOME MEASURES--Intelligence quotient as measured by the AH4 test and amount of decline in cognitive function with age as estimated by the difference between score on the Mill Hill vocabulary test and score on the AH4 test. RESULTS--Score on the intelligence test was higher in people who had a large biparietal head diameter at birth, but it was not related to any other measure of body size or proportions. No association was found between decline in cognitive function and any measure of size or proportions at birth. CONCLUSION--Impaired fetal growth was not associated with poorer cognitive performance in adult life. Adaptations made by the fetus in response to conditions that retard its growth seem to be largely successful in maintaining brain development.     

Sister-chromatid exchanges in human lymphocytes exposed during Go to four classes of DNA-damaging chemicals.

Human lymphocytes were treated prior to mitogenic stimulation with varying concentrations of 6 cytostatic drugs representing 4 classes of DNA-damaging chemicals. Afterwards the cells were washed to remove residual chemical and cultured in the presence of bromodeoxyuridine for analysis of sister-chromatid exchanges (SCEs). A dose-related increase in SCEs was observed in cells exposed during Go to the alkylating chemicals mitomycin C, chlorambucil, and thiotepa, while significant increases in SCEs were not noted in cultures exposed to methotrexate, cytarabine, or bleomycin. These findings suggest that not all classes of clatogenic chemicals which induce SCEs in proliferative cells substituted with BUdR are capable of inducing long-lived lesions in the DNA of Go lymphocytes that can lead to SCE formation.     

Embryonic and larval development of brown trout, Salmo trutta L.: exposure to aluminium, copper, lead or zinc in soft, acid water

Freshly fertilized ova, eyed ova and yolk-sac fry of brown trout, Salmo trutta L., were exposed to each of four trace metals (aluminium: 6000 nmol l^?1; copper: 80 nmol l^?1; lead: 50 nmol l^?1; zinc: 300 nmol l^?1) while held in flowing artificial soft-water media maintained at pH 4.5 or 5.6 and [Ca] 20 or 200 μmol l^?1. In continuous exposure from fertilization, survival of ova was severely affected at pH 4.5 and [Ca] 20 μmol l^?1, regardless of the presence of Cu, Pb or Zn; Al reduced embryonic mortality and improved hatching success. High ambient [Ca] at pH 4.5 increased egg survival. At ‘swim-up’, surviving fry exposed to Al or Pb had lower whole body Ca, Na and K content, irrespective of pH or ambient [Ca]. Cu reduced whole body Ca and K content at pH 5.6 and [Ca] 200 μmol^?1, and whole body Ca, Na and K content in the other media. Zn reduced whole body mineral content at pH 5.6 and [Ca] 20 μmol l^?1. Whole body Mg content was reduced by all trace metals at pH 5.6 and [Ca] 20 μmol l^?1, and by Cu at pH 5.6 and [Ca] 200 μmol l^?1. Al and Cu impaired skeletal calcification at pH 5.6 at both ambient [Ca]; Pb only at [Ca] 20 μmol I^?1. Zn enhanced calcification at pH 4.5 and [Ca] 200 μmol l^?1. In the absence of trace metals, low pH reduced body Ca, Na, K content and skeletal calcification at [Ca] 200 μmol l^?1. The uptake of Ca, Na and K, measured at regular intervals from hatching was impaired to the same extent by all treatments at pH 4.5, irrespective of ambient [Ca] or trace metal presence. At pH 5.6, irrespective of ambient [Ca], Al, Cu and Pb impaired Ca and K uptake. The rate of Na uptake was reduced by Al and Cu. Al-treated yolk-sac fry, exposed to low ambient [Ca] from 200–300° days post-hatch, suffered high mortalities regardless of pH. Ca, Na and K uptake was impaired by all treatments at pH 4.5, and by Al and Cu at pH 5.6 in a similar exposure period. The development of the early stages of brown trout in the presence of trace metals is discussed in relation to recruitment failure in areas of soft, acid water.