Dynamic organization of mitochondrial membranes: A crosslinking study with dimethylsuberimidate

Rat liver mitochondria were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effects were evaluated kinetically. Concurrently with the modification of amino groups, mitochondrial proteins were crosslinked and the organelles lost their osmotic response. When the dimethylsuberimidate reaction was performed in the presence of succinate, more primary amino groups were available when compared with a sucrose medium. Concomitantly, osmotic stabilization and crosslinking of mitochondrial proteins were accelerated. The activity of aspartate aminotransferase was also studied in crosslinked mitochondria. The enzyme activity was only slightly modified when mitochondria were amidinated in a sucrose medium and solubilized thereafter with Triton X-100 or cetyltrimethylammonium bromide. In contrast, in the presence of succinate, 60% of activity was lost after solubilization with Triton X-100, but not after solubilization with cetyltrimethylammonium bromide. This finding was correlated with the changes in intramitochondrial localization of the enzyme (A. Waksman and A. Rendon, 1974,Biochimie54, 907–924). When carbonylcyanide-p-trifluoromethoxyphenylhydrazone was added in both cases (sucrose or sucrose plus succinate), the rates of osmotic stabilization, amidination reaction, crosslinking of proteins, and aspartate aminotransferase activity were similar to those observed in a sucrose medium alone. The present results suggest that organizational changes of the mitochondrial membranes induced by succinate, including intramitochondrial protein movement, are prevented by carbonylcyanide-p-trifluoromethoxyphenylhydrazone.     

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology

Summary This is the third in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxribonucleate)-3-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article describes the structure of the nuclease and of a nuclease-inhibitor complex as determined by x-ray crystallography. The crystal structures are correlated with some of the known chemical and enzymological properties of the enzyme, and the three areas combined to propose a mechanism of action.This article is the third in a series of four devoted to the stapholoccal nuclease. Reviews concerning its isolation and enzymology (1) as well as the features of its ligand binding site (2) have appeared in previous issues. Work from this laboratory has been supported by grants from the National Institute of General Medical Sciences, NIH and from the Robert A. Welch Foundation to F. A. Cotton and E. E. Hazen, Jr.     

Evaluation of Bottlenecks in the Late Stages of Protein Secretion in Bacillus subtilis

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis α-amylase (AmyL), Escherichia coli TEM β-lactamase (Bla), human pancreatic α-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.     

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.     

Encoding of sound duration by neurons in the auditory cortex of the little brown bat, Myotis lucifugus

Responses of 117 single- or multi-units in the auditory cortex (AC) of bats (Myotis lucifugus) to tone bursts of different stimulus durations (1– 400 ms) were studied over a wide range of stimulus intensities to determine how stimulus duration is represented in the AC. 36% of AC neurons responded more strongly to short stimulus durations showing short-pass duration response functions, 31% responded equally to all pulse durations (i.e., all-pass), 18% responded preferentially to stimuli having longer durations (i.e., long-pass), and 15% responded to a narrow range of stimulus durations (i.e., band-pass). Neurons showing long-pass and short-pass duration response functions were narrowly distributed within two horizontal slabs of the cortex, over the rostrocaudal extent of the AC. The effects of stimulus level on duration selectivity were evaluated for 17 AC neurons. For 65% of these units, an increase in stimulus intensity resulted in a progressive decrease in the best duration. In light of the unusual intensity-dependent duration responses of AC neurons, we hypothesized that the response selectivities of AC neurons is different from that in the brainstem. This hypothesis was validated by results of study of the duration response characteristics of single neurons in the inferior colliculus. Accepted: 8 November 1996