Characterisation of the Bax-nucleophosmin interaction: the importance of the Bax C-terminus

The molecular chaperone nucleophosmin has been identified as a novel Bax binding protein with this interaction proposed to be a key event in the activation and translocation of Bax in mitochondrial dysfunction and apoptotic cell death. Using a proximity assay, we have quantitatively defined the high affinity and saturable interaction between Bax and nucleophosmin indicative of a competitive and specific mechanism. Binding of full length Bax to nucleophosmin was only observed after conformational change was induced using non-ionic detergents (e.g., NP-40). The Bax-nucleophosmin interaction was inhibited by a Bax C-terminal antibody (IC₅₀ = 1 nM) but minimally affected by antibodies directed against either the N-terminus or α-helices 4 and 5. Bcl-2 and p53 inhibited the interaction between full length activated Bax and nucleophosmin. The proximity assay based on the Bax-nucleophosmin interaction was robust and reproducible (Z′ = 0.50) facilitating its use for screening a small chemical library. A low molecular weight non-peptide compound, 2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide, partially inhibited the Bax-nucleophosmin interaction (IC₅₀ = 100 nM) and also attenuated UV-induced cell death of HEK293 cells. The present investigations demonstrate the importance of exposure of the C-terminus of Bax for its interaction with nucleophosmin. These protein–protein interaction assays provide a technical approach both for the study of Bax-interacting proteins and for the discovery of novel anti-apoptotic agents. L. Kerr and J. Sharkey have contributed equally to this work.     

Under pressure: the search for the essential mechanisms of hypertension

High blood pressure, or hypertension, is a very common disorder with a substantial impact on public health because of its associated complications. Despite the high prevalence of essential hypertension and years of research, the basic causes remain obscure. Here I review recent advances in understanding the pathophysiology of hypertension. I present a general overview of the field and, by necessity, use broad strokes to portray recent progress and place it in context. For this purpose, I use illustrative examples from the large number of important developments in hypertension research over the last five years. The intent of this review is to provide a sense of where the field is progressing, with an emphasis on work that sheds light on pathogenic mechanisms and that is therefore likely to inform new translational advances.     

beta-Endorphin: analgesic and receptor binding activity of non-mammalian homologs

Analgesic potencies of turkey, ostrich and des-acetyl salmon beta-endorphins have been measured in the tail-flick test and binding affinities determined by radio-receptor assay. The duration of analgesia and the slope of the dose-response curves generated by these peptides are similar to those elicited by mammalian beta-endorphins. This suggests that they act in vivo and in vitro on the same population of opiate receptors. The ratio of binding to analgesic potencies observed for these peptides varies nearly sixfold. Structure-activity analysis suggests that a basic side-chain at position 9 is required in order to produce a high opiate activity both in vivo and in vitro. A reexamination of the biological activities of camel beta-endorphin shows that the analgesic potency and binding affinity of this peptide are respectively 171 and 2.7 times higher than human beta-endorphin. His-27 and/or Gln-31 may contribute to this increased potency. The dissociation of radioreceptor binding affinity from analgesic potency in these naturally occurring beta-endorphin homologs suggests that either the conditions under which the binding assay is performed mask the true binding potency in the brain or that, once bound to the appropriate receptor, these homologs do not possess equal ability to produce biological effects.     

The importance of being essential: EMBRYO-DEFECTIVE genes in Arabidopsis

With the completion of the sequence of the first bacterial genomes, scientists have been able to address the question: How many genes are required for cell viability? In attempting to reply to this question, the concept of the minimal gene set was developed and validated by systematic gene disruption. In a similar manner, whole genome comparisons and systematic Knock-Out have been performed in eukaryotes and have led to the identification to date of the set of essential genes in yeast and C. elegans. In the plant kingdom, the sequence of the Arabidopsis genome together with large-scale functional genomics programs now allow us to address the question of essentiality in Arabidopsis. These concerted efforts have resulted in the identification to date of up to 219 genes essential for seed development (EMBRYO-DEFECTIVE, EMB, genes). With this basic knowledge, we can start a valid comparison of essentiality in Arabidopsis and in other eukaryotes based on functional categories and orthologous relationships. Furthermore, the function of the EMB genes in the particular context of eukaryote evolution driven by whole genome duplications and selective gene loss will be discussed.     

Biofeedback treatments of essential hypertension

Fifteen years of research in the self-regulatory treatment of hypertension by the author is summarized. A model relating expectations, task performance, home practice, and biochemical variables to the thermal biofeedback treatment of hypertension is presented.     

Biofeedback treatments of essential hypertension

Fifteen years of research in the self-regulatory treatment of hypertension by the author is summarized. A model relating expectations, task performance, home practice, and biochemical variables to the thermal biofeedback treatment of hypertension is presented.Essentially, all of the research reported herein was supported by various grants from NHLBI: HL-14906, HL-18814, HL-27622, and HL-31189.     

beta-Endorphin: hyperthermia in mice by intravenous injection

Effects of intravenous beta-endorphin on body temperature and body weight loss were studied in naive and morphine-dependent mice. beta-Endorphin at doses 2.6-25.5 mg/kg injected intravenously caused hyperthermia in naive mice as well as in morphine-dependent mice. In addition, beta-endorphin and morphine reduced body-weight loss during the morphine withdrawal.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

beta-Endorphin in human plasma: basal and pathologically elevated levels.

β-Endorphin-like immunoreactivity was measured in plasma of normal human subjects and in plasma of patients with pathologically elevated ACTH levels. The antiserum used displayed the same avidity for human β-endorphin and human β-lipotropin (detection limit for both peptides 1–2 fmoles/tube). Gel chromatography of the immunoreactive components in plasma of normal subjects indicated the presence of both β-lipotropin (2.1 -10.1 fmoles/ml) and β-endorphin (3.5–6.4 fmoles/ml). A close correlation between immunoreactive β-endorphin and ACTH was found in plasma of patients suffering from Addison's disease, Cushing's disease and exhibiting Nelson's syndrome. Elevated levels of β-endorphin-like immunoreactivity in plasma of these patients were due to both β-lipotropin and β-endorphin.     

Phenytoin-valproate interaction: importance of saliva monitoring in epilepsy.

Sodium valproate is often used with phenytoin when epilepsy cannot be controlled by a single drug. Sodium valproate depresses phenytoin protein binding and so invalidates plasma phenytoin monitoring as a means of determining precise phenytoin dosage requirements. Plasma and saliva phenytoin and plasma valproate concentrations were measured in 42 patients with epilepsy receiving both drugs. Phenytoin protein binding was also measured by ultrafiltration in 19 of these patients and 19 patients taking phenytoin alone. Saliva phenytoin concentration bore the same close correlation to unbound (therapeutically active) phenytoin in patients receiving both drugs as it did in patients receiving phenytoin alone, whereas plasma total phenytoin did not. The same therapeutic range for saliva phenytoin (4-9 mumol/1; 1-2 microgram/ml) was therefore valid in both groups. The depression of phenytoin binding was directly related to the plasma concentration of valproate both in random samples taken from the 42 patients and in samples taken throughout the day in two of these patients. This was confirmed in vitro. Even when the concentration of valproate is known the degree of binding cannot be predicted. Saliva rather than plasma monitoring of phenytoin treatment is therefore valuable in the presence of valproate and with reduced phenytoin binding from any cause.     

The genetic dissection of essential hypertension

QTL mapping in humans and rats has identified hundreds of blood-pressure-related phenotypes and genomic regions; the next daunting task is gene identification and validation. The development of novel rat model systems that mimic many elements of the human disease, coupled with advances in the genomic and informatic infrastructure for rats, promise to revolutionize the hunt for genes that determine susceptibility to hypertension. Furthermore, methods are evolving that should enable the identification of candidate genes in human populations. Together with the computational reconstruction of regulatory networks, these methods provide opportunities to significantly advance our understanding of the underlying aetiology of hypertension.     

Aldosterone and renin in essential hypertension.

A review of some recent laboratory findings indicates definite disturbances in aldosterone metabolism and regulation in patients with mild essential hypertension: (a) a significant mean increase in plasma aldosterone concentration in patients with mild and stable essential hypertension, in contrast to the absence of any difference in patients with labile borderline essential hypertension when in a normotensive phase, compared with control subjects; and (b) a significant mean decrease in metabolic clearance rate of aldosterone, associated with a 12% decrease in hepatic blood flow and an increased binding of aldosterone to a transcortin-like plasma globulin. The secretion rate of 18-hydroxy-11-deoxycorticosterone is above the upper range of normal in 60% of patients with mild, uncomplicated essential hypertension. The incidence of low-renin hypertension, when age and race are taken into account, is much lower than previously assumed. Unless measurements are repeated over a long period, one or two low values of plasma renin cannot be considered a permanent marker indicating a special category of patients with essential hypertension. Tonin, a new enzyme discovered by Boucher, which forms angiotensin II directly from a plasma protein, from the tetradecapeptide substrate and from angiotensin I, is present in most tissues, but in highest concentration in the submaxillary gland. This enzyme is under the control of beta-adrenergic receptors.     

Aldosterone and magnesium in essential arterial hypertension

Thirty mildly hypertensive patients and 27 patients with severe essential hypertension and high levels of aldosterone were selected for a study of the relationship between plasma aldosterone and magnesium in essential arterial hypertension; levels of calcium and potassium were also studied. Thirty-six individuals were used as a control group. Our findings indicate that as plasma aldosterone levels increase, serum magnesium levels decrease correspondingly: in mild hypertensives with low levels of plasma aldosterone p less than 0.05 and in the most severely hypertensive patients with high levels of plasma aldosterone p less than 0.001. In this latter group we also found an inverse correlation between serum magnesium and systolic arterial pressure (p less than 0.001) and diastolic pressure (p less than 0.01). In these patients a significant increase in urinary excretion of magnesium was found, with levels 3 times higher than in the control group. These findings suggest a close relationship between changes in plasma aldosterone and magnesium. Possibly the aldosterone contributes through this mechanism to maintaining the hypertensive state in essential arterial hypertension. This action is exercised directly through the kidney, leading to a small but constant urinary loss of magnesium. This in turn leads to a chronic depletion of magnesium in hypertensives who have high levels of plasma renin activity and highly elevated plasma aldosterone.