Proton translocation in cytochrome-deficient mutants of Escherichia coli.

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.     

The measurement of partial specific volumes and sedimentation coefficients by sucrose density centrifugation

The method of Martin and Ames (1961, J. Biol. Chem.236, 1372) gives good estimates of the s_20,w of proteins when the SW40 rotor is used with a sucrose gradient. Viscosities of sucrose in D₂O were measured, and the data were used in computer simulations to test alternate approaches to estimating $\text{v}$ and s_20,w values by comparisons with standards. The method of Meunier et al. (1972, FEBS Lett.24, 63) for $\text{v}$ was shown to be optimal. For s_20,w estimations, substantial errors were found with the methods of Bon et al. (1973, Eur. J. Blochem.35, 372) and especially Meunier et al. When standards and unknowns have the same $\text{v}$, and the gradient is made up in water or dilute buffer, the simple ratio method of Martin and Ames gives most accurate results for s_20,w. For all other cases, an alternative procedure is described.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

The origin of the Adams-Schuster difference spectrum of oxyhemoglobin.

The characteristic difference spectrum reported by Adams and Schuster (Biochem. Biophys. Res. Commun. 1974, $\text{58}$, 525) on the addition of inositol hexaphosphate to oxyhemoglobin is similar to the difference spectrum between (i) isolated α- and β-chains, (ii) α- and β-semihemoglobins, (iii) addition of inorganic phosphate to oxyhemoglobin, (v) change in temperature of a solution of oxyhemoglobin, (v) change in pH of carp carboxyhemoglobin and (vi) addition of inositol hexaphosphate to α-semihemoglobin. The spectrum may also be generated by differentiation of the spectra of oxyhemoglobin and carboxyhemoglobin, implying that the common feature of the results reported above is a shift in the position of the absorption bands. This shift may arise from several causes and so its interpretation is uncertain.     

Mu-induced polarity in the Escherichia coli K-12 ent gene cluster: evidence for a gene (entG) involved in the biosynthesis of enterochelin.

A strain of Escherichia coli K-12 has been isolated that carries a Mu bacteriophage-induced mutation in the ent gene cluster. Nutritional tests together with examination of the compounds accumulated by the mutant strain indicated that the mutant was blocked both in the synthesis of 2,3-dihydroxy-benzoate and its subsequent conversion into enterochelin. Enzymic complementation assays of the mutant with several mutants each affected in one of the ent genes showed that the Mu-induced mutant was entA-, entB-, entC+, entD+, entE+, and entF+. Since the mutant produced the entD, entE, and entF gene products but was unable to produce enterochelin from 2,3-dihydroxybenzoate, it must therefore be affected in an additional protein concerned with this conversion. It is therefore postulated that the Mu-induced mutation affects a previously unrecognized gene, entG. Genetic experiments indicate that the mutation in strain AN462 which affects the three ent genes is the result of a single insertion of Mu in the ent gene cluster. This polarity mutant therefore provides evidence that three of the ent genes are part of an operon.     

Biochemical studies on the esterase enzymes of four species of nemertean worms

Esterase enzymes were studied biochemically in extracts of four species of nemertean worms. Optimal enzymic activity occurs within the range pH 6.0–8.2. The relative amounts of esterolytic activity differ between species and, within individual species, between pH optima. It is possible that these differences may, at least in part, be related both to phylogeny and the pattern of digestive physiology.10⁻² M sodium taurocholate and 10⁻³ M lead nitrate possess mainly inhibitory effects, whereas 10⁻³ M cysteine hydrochloride functions predominantly as an activator. The precise effect in each case depends both upon the species and the pH of incubation.Esterases at pH 7.4 are most active at temperatures within the range 40–51 °C, depending upon the species concerned.     

On the occurrence of males and production of ephippial eggs in populations of Daphniopsis studeri (Cladocera) in lakes of the Vestfold and Larsemann Hills, East Antarctica

Populations of the cladoceran Daphniopsis studeri Rühe in freshwater and brackish lakes of eastern Antarctica have been thought to consist solely of females that reproduce parthenogenetically by the production of ameiotic subitaneous eggs. This note reports the presence of male D. studeri and the production of ephippial (sexual) eggs in a number of lakes of the Vestfold and Larsemann Hills, which indicate the possibility of sexual reproduction within these populations. Received: 12 May 1997 / Accepted: 8 September 1997     

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.