The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element. The ␣-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions. The von Hippel-Lindau protein (pVHL) mediates the ubiquitination and rapid degradation of HIF-␣ (including HIF-1␣ and HIF-2␣). Post-translational hydroxylation of a proline residue in the oxygen-dependent degradation (ODD) domain of HIF-␣ is required for the interaction between HIF and VHL. It has previously been established that cobalt mimics hypoxia and causes accumulation of HIF-1␣ and HIF-2␣. However, little is known about the mechanism by which this occurs. In an earlier study, we demonstrated that cobalt binds directly to the ODD domain of HIF-2␣. Here we provide the first evidence that cobalt inhibits pVHL binding to HIF-␣ even when HIF-␣ is hydroxylated. Deletion of 17 amino acids within the ODD domain of HIF-2␣ that are required for pVHL binding prevented the binding of cobalt and stabilized HIF-2␣ during normoxia. These findings show that cobalt mimics hypoxia, at least in part, by occupying the VHL-binding domain of HIF-␣ and thereby preventing the degradation of HIF-␣.Hypoxia is a critical stimulus in many physiological and disease states (1). Cells respond to hypoxia by regulating the expression of a number of genes, including erythropoietin, vascular endothelial growth factor, and various glycolytic enzymes (2-5). This regulation is mediated in part by transcription factors of the hypoxia-inducible factor (HIF) 1 family (6). HIF-1␣ and HIF-2␣ are basic helix-loop-helix Per-Arnt-Sim (PAS) domain proteins (7) that form a heterodimer with the aryl hydrocarbon nuclear receptor translocator protein. Previous studies have shown that HIF-1␣ protein accumulates rapidly during hypoxia without a significant increase in HIF-1␣ mRNA levels (8). HIF-2␣, which is also known as endothelial PAS domain protein-1, shares close sequence and structural homology with HIF-1␣ (9). Like HIF-1␣, the levels of HIF-2␣ protein are low during normoxia and accumulate when cells are exposed to hypoxia, proteasomal inhibitors, transition metals (e.g. cobalt), iron chelators, or reducing agents (10). During normoxia, the HIF-␣ (HIF-1␣ and HIF-2␣ are referred to here simply as HIF-␣, except where noted otherwise) proteins are continuously degraded by ubiquitin-and proteasome-dependent pathway. Detailed studies of HIF-␣ proteins revealed a 200-amino acid sequence, called the oxygen-dependent degradation domain (ODD) that is responsible for its degradation in the presence of oxygen (11,12). The von Hippel-Lindau (pVHL) protein, a tumor suppressor protein, mediates the ubiquitination and degradation of HIF-␣ by binding to the ODD domain under conditions of normoxia (13,14). Recent findings revealed that pVHL-mediated degradation requires hydroxylation of specific proline residues within the ODD (15-18). The hydroxylation of these proline residues may be critical for regulating the HI...

Though the ischemic penumbra has been classically described on the basis of blood flow and physiologic parameters, a variety of ischemic penumbras can be described in molecular terms. Apoptosis-related genes induced after focal ischemia may contribute to cell death in the core and the selective cell death adjacent to an infarct. The HSP70 heat shock protein is induced in glia at the edges of an infarct and in neurons often at some distance from the infarct. HSP70 proteins are induced in cells in response to denatured proteins that occur as a result of temporary energy failure. Hypoxia-inducible factor (HIF) is also induced after focal ischemia in regions that can extend beyond the HSP70 induction. The region of HIF induction is proposed to represent the areas of decreased cerebral blood flow and decreased oxygen delivery. Immediate early genes are induced in cortex, hippocampus, thalamus, and other brain regions. These distant changes in gene expression occur because of ischemia-induced spreading depression or depolarization and could contribute to plastic changes in brain after stroke.

Unanesthetized decorticate cats walked or ran normally on a treadmill either spontaneously or during electrical stimulation of the subthalamic "locomotor" region. The respiratory response usually preceded the locomotor response and increased in proportion to locomotor activity despite control or ablation of respiratory feedback mechanisms. Respiration increased similarly in paralyzed animals during fictive locomotion despite the absence of muscular contraction or movement. Hypothalamic command signals are thus primarily responsible for the proportional driving of locomotion and respiration during exercise.

A complete understanding of the neural mechanisms responsible for the chemoreceptor and baroreceptor reflexes requires precise knowledge of the locations and chemical phenotypes of higher-order neurons within these reflex pathways. In the present study, the protein product (Fos) of the c-fos protooncogene was used as a metabolic marker to trace central neural pathways following activation of carotid sinus nerve afferent fibers. In addition, immunohistochemical double-labeling techniques were used to define the chemical phenotypes of activated neurons. Both electrical stimulation of the carotid sinus nerve and physiological stimulation of the carotid bodies by hypoxia induced Fos-like immunoreactivity in catecholaminergic neurons containing tyrosine hydroxylase or phenylethanolamine-N-methyltransferase in the ventrolateral medulla oblongata and, to a lesser degree, in the dorsal vagal complex. Tyrosine hydroxylase/Fos colocalization was also observed in the locus coeruleus and the A5 noradrenergic cell group in pons. Many serotoninergic neurons in nucleus raphe pallidus, nucleus raphe magnus, and along the ventral medullary surface contained Fos-like immunoreactivity. In pons and midbrain, Fos-like immunoreactivity was observed in the lateral parabrachial and Kölliker-Fuse nuclei, the inferior colliculus, the cuneiform nucleus, and in the vicinity of the Edinger-Westphal nucleus, but no catecholaminergic or serotoninergic colocalization was observed in these regions. Although Fos-labeled cells were observed within and lateral to the dorsal raphe nucleus, few were catecholaminergic or serotoninergic. This study further defines a potential central neuroanatomical substrate for the chemoreceptor and/or baroreceptor reflexes.

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