Performance monitoring by the supplementary eye field

Intelligent behaviour requires self-control based on the consequences of actions. The countermanding task is designed to study self-control; it requires subjects to withhold planned movements in response to an imperative stop signal, which they can do with varying success. In humans, the medial frontal cortex has been implicated in the supervisory control of action. In monkeys, the supplementary eye field in the dorsomedial frontal cortex is involved in producing eye movements, but its precise function has not been clarified. To investigate the role of the supplementary eye field in the control of eye movements, we recorded neural activity in macaque monkeys trained to perform an eye movement countermanding task. Distinct groups of neurons were active after errors, after successful withholding of a partially prepared movement, or in association with reinforcement. These three forms of activation could not be explained by sensory or motor factors. Our results lead us to put forward the hypothesis that the supplementary eye field contributes to monitoring the context and consequences of eye movements.     

Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain

The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.     

MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines

The product of the MDR1 gene (P-gp) has been implicated in the transport of cholesterol from plasma membrane to endoplasmic reticulum for esterification. In previous studies on leukemia cell lines, we suggested that cholesterol esterification may regulate the rate of cell growth and that the MDR1 gene might be involved in this process by modulating intracellular cholesterol esters levels. To further investigate this matter, the rate of cell growth, cholesterol metabolism, expression of the MDR1 gene, and P-gp activity were compared in KB cell lines displaying differences in expression and function of P-gp (drug-sensitive phenotype versus MDR phenotype). The rate of cell growth correlated with cholesterol esterification in all KB cell lines, whereas the over-expression of MDR1 observed in the MDR cell lines was not always associated with an increased capacity of cells to esterify cholesterol. Two known inhibitors of P-gp activity, progesterone and verapamil, strongly inhibited both cholesterol esterification and cell proliferation in all KB cell lines, but they affected intracellular accumulation of labeled vinblastine only in MDR cell lines. These results further support a role for cholesterol esters in the regulation of cell growth and suggest that the P-gp expressed in MDR KB cells is not involved in the general process leading to cholesterol esterification. Received 14 February 2000; received after revision 10 April 2000; accepted 8 May 2000     

New insights into copper monooxygenases and peptide amidation: structure, mechanism and function

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.     

Expression and cellular distribution of perchloric acid-soluble protein is dependent on the cell-proliferating states of NRK-52E cells

To clarify the biological role of kidney perchloric acid-soluble protein 1 (K-PSP1), its expression and intracellular distribution were examined in normal rat kidney epithelial NRK-52E cells. K-PSP1 expression was low during the proliferating phase and high in the stationary phase, and shown to have a negative relationship with the protein-synthesizing activity of the cells. Immunocytochemical studies revealed that K-PSP1 is predominantly located in the cytosol, especially in endoplasmic reticulum and Golgi apparatus of proliferating cells. In the stationary phase, K-PSP1 was not detected immunologically even though protein and mRNA expression were high. This disappearance of reactivity with anti-serum seems to be due to a conformational change in K-PSP1 induced by unknown factors. These results suggest that the role of K-PSP1 is to regulate cell proliferation, and this may be related to a previously reported ability to inhibit protein synthesis.     

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons

Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent major dementia-associated abnormalities in Alzheimer’s disease (AD). This study examined the role of oxidative stress as a factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated with H₂O₂ (9–90 μM) or desferrioxamine (2–25 μM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function, and pro-apoptosis and sprouting gene expression. H₂O₂ treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The H₂O₂-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin, and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection), and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast, hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD, both oxidative stress and hypoxic injury can contribute to AD neurodegeneration. Received 24 May 2000; received after revision 7 July 2000; accepted 27 July 2000     

The amino acid sequence of the agglutinin isolated from the red marine alga Bryothamnion triquetrum defines a novel lectin structure

The primary structure of a lectin isolated from the red alga Bryothamnion triquetrum was established by combination of Edman degradation of sets of overlapping peptides and mass spectrometry. It contains 91 amino acids and two disulphide bonds. The primary structure of the B. triquetrum lectin does not show amino acid sequence similarity with known plant and animal lectin structures. Hence, this protein may be the paradigm of a novel lectin family.     

Differential basal synthesis of Hsp70/Hsc70 contributes to interindividual variation in Hsp70/Hsc70 inducibility

The source of intraspecies variation in the expression of heat shock proteins (HSPs) remains unresolved but could shed light on differential stress tolerance and disease susceptibility. This study investigated the influence of variable basal HSP synthesis on differential inducibility of HSP synthesis. Basal and heat-induced synthesis of the major HSP families in peripheral blood monocytes from healthy donors (n=42) were analysed using biometabolic labelling and densitometry. Basal Hsp70/Hsc70 synthesis and percentage induction of Hsp70/Hsc70 synthesis were significantly correlated (r=−0.57, p<0.0001), and described most accurately by an exponential decay equation (R=0.68, R²=0.46). This regression equation suggests that increasing levels of basal Hsp70/Hsc70 synthesis are accompanied byan exponential decrease in the percentage induction of Hsp70/Hsc70 synthesis. The model fits data from European and non-European population groups independently, although both coefficients in the regression equation were larger for non-Europeans. This implies population group as an additional factor influencing differential HSP expression. The differential inducibility of Hsp70/Hsc70 due to variable basal synthesis of Hsp70/Hsc70 and based upon population group may contribute to differential stress tolerance or disease susceptibility. Received 27 March 2000; received after revision 19 June 2000; accepted 20 June 2000     

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease

The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ _1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H₂O₂ modification of Aβ _1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ _1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease Received 26 September 2000; accepted 26 September 2000