The effect of chronic hypobaric hypoxia (28 days, 455 Torr) on the organization of brain vessels was studied in Balb/c mice. In comparison to age-matched controls kept at sea level, emulsion-perfused capillaries in hypoxic mice showed marked dilation in all brain areas studied. Capillary length per unit volume of tissue (Lv) was increased in the cerebellar granular layer, the caudate nucleus, the globus pallidus, the substantia nigra, the superior colliculus, and the dentate gyrus. There was a selective increase of Lv in the hippocampus (CA1 strata pyramidale and lacunosum and CA3 strata pyramidale and oriens) and in somatosensory cortex layers V and VI, motor cortex layers II, III, V, and VI, and auditory cortex layers II and III. An increase in capillary surface area per unit volume of tissue was also determined in several brain areas, including layer IV of somatosensory cortex, where Lv was not significantly increased. The O2 diffusion conductance and PO2 in the tissues were estimated with a mathematical model. The remodeling of capillary diameter and length during chronic hypoxia accounts for the significant increase of O2 conductance to neural tissues. Also the estimated tissue PO2 in chronic brain hypoxia is markedly increased in the caudate nucleus and the substantia nigra compared with acute hypoxia. These results suggest that formation of new capillaries is an important mechanism to restore the O2 deficit in chronic brain hypoxia and that local rates of energy utilization may influence angiogenesis in different areas of the brain.

It is a common experience to sacrifice sleep to meet the demands of our 24-h society. Current estimates reveal that as a society, we sleep on average 2 h less than we did 40 years ago. This level of sleep restriction results in negative health outcomes and is sufficient to produce cognitive deficits and reduced attention and is associated with increased risk for traffic and occupational accidents. Unfortunately, there is no simple quantifiable marker that can detect an individual who is excessively sleepy before adverse outcomes become evident. To address this issue, we have developed a simple and effective strategy for identifying biomarkers of sleepiness by using genetic and pharmacological tools that dissociate sleep drive from wake time in the model organism Drosophila melanogaster. These studies have identified a biomarker, Amylase, that is highly correlated with sleep drive. More importantly, both salivary Amylase activity and mRNA levels are also responsive to extended waking in humans. These data indicate that the fly is relevant for human sleep research and represents a first step in developing an effective method for detecting sleepiness in vulnerable populations.Drosophila ͉ saliva ͉ sleep deprivation I t has been suggested that forced and self-inflicted sleep loss have reached epidemic proportions in Western industrialized populations (1, 2), costing billions of dollars in lost productivity and creating hazardous conditions on our roadways (3), in our skies (4), and in our hospitals (5). The National Highway Traffic Safety Administration estimates that 20% of motor vehicle crashes are attributed to sleepiness and fully 37% of adult drivers report falling asleep at the wheel at some point in their lives. Moreover, both regional and long-haul pilots accumulate sleep debt during trips, fall asleep in the cockpit, and experience levels of sleepiness that are associated with performance decrements (6, 7). In the hospital setting, training demands frequently disrupt the sleep of medical residents, which is then associated with increased attentional failures and medical errors (8). Indeed, after a heavy call rotation, the driving performance of medical residents was similar to those with a blood alcohol level of 0.05 g % (9) and is associated with increased risk of falling asleep while driving (10).Given the magnitude of this problem, it is not surprising that the sleep community, public health officials, and others have devoted considerable attention toward minimizing the negative impact of sleep loss on public health and safety (11). In addition to more focused basic research and increased educational campaigns to create public awareness, both regulatory and legislative initiatives have been implemented to address this problem. A general theme that has emerged from all of these efforts has been the importance of identifying a simple and quantifiable biomarker of sleepiness (11)(12)(13). A biomarker of sleepiness should be responsive to increasing levels of sleep debt and should only be activated by period...

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation. Sarcomeric mitochondrial creatine kinase (sMtCK)1 is a member of a gene family of four homologous CK genes (1-4). The M-CK and B-CK genes encode soluble, cytoplasmic homodimeric enzymes necessary for ATP production from creatine phosphate at sites of high energy utilization, such as the contractile apparatus. These cytosolic CKs share about 85% similarity to each other in a particular species, and each has 77-91% homology among vertebrate species. Two additional genes encode different mitochondrial CKs, which are localized on the outer surface of the inner mitochondrial membrane (IMM) (2, 3). sMtCK expression is restricted to heart and skeletal muscle, but ubiquitous MtCK is present in many tissues, especially brain and smooth muscle. Mammalian MtCKs are only 60 -65% similar to cytosolic CKs and about 80% homologous to each other, but sMtCKs share 95% identity across species. This suggests that, in addition to enzymatic functions, ubiquitous MtCK and sMtCK may have slightly different structural roles requiring sequence conservation across species.We previously isolated and characterized cDNA clones encoding human (2, 3) and rat (7) MtCKs. The rat sMtCK cDNA coding region consists of 1260 nucleotides, and the first 117 bp encode the transit peptide, responsible for transport of de novo synthesized precursor sMtCK from cytosol into mitochondria. During this translocation of pre-sMtCK to the intermembranous space, the transit peptide is proteolytically removed, allowing formation of the active, mature subunit (2, 4).Both MtCK proteins can f...

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