Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia

Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t(1/2) was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O(2)/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.     

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.     

Temporal patterns of visitation of birds and mammals at mineral licks in the Peruvian Amazon

Mineral licks are key ecological resources for many species of birds and mammals in Amazonia, providing essential dietary nutrients and clays, yet little is known about which species visit and their behaviors at the mineral licks. Studying visitation and behavior at mineral licks can provide insight into the lives of otherwise secretive and elusive species. We assessed which species visited mineral licks, when they visited, and whether visits and the probability of recording groups at mineral licks were seasonal or related to the lunar cycle. We camera trapped at 52 mineral licks in the northeastern Peruvian Amazon and detected 20 mammal and 13 bird species over 6,255 camera nights. Generalized linear models assessed visitation patterns and records of groups in association with seasonality and the lunar cycle. We report nocturnal curassows (Nothocrax urumutum) visiting mineral licks for the first time. We found seasonal trends in visitation for the black agouti (Dasyprocta fuliginosa), red howler monkey (Alouatta seniculus), blue‐throated piping guan (Pipile cumanensis), red brocket deer (Mazama americana), collared peccary (Pecari tajacu), and tapir (Tapirus terrestris). Lunar trends in visitation occurred for the paca (Cuniculus paca), Brazilian porcupine (Coendou prehensilis), and red brocket deer. The probability of recording groups (>1 individual) at mineral licks was seasonal and related to lunar brightness for tapir. Overall, our results provide important context for how elusive species of birds and mammals interact with these key ecological resources on a landscape scale. The ecological importance of mineral licks for these species can provide context to seasonal changes in species occupancy and movement.

Many species of animals in the Amazon visit mineral licks, consuming soil to supplement their diet. Using camera trap data, we show that visits to mineral licks are seasonal for many species. These trends in visitation are likely due to species‐specific factors such as reproduction, predator avoidance, seasonal diet shifts, and resource use.     

How do oncoprotein mutations rewire protein–protein interaction networks?

The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein–protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein–protein interaction networks and drive the cancer phenotype.     

Ovarian functionality in Poeppig's woolly monkey (Lagothrix poeppigii)

This study examined ovarian features of 60 Poeppig's woolly monkey females in different reproductive stages, collected from wild animals hunted by rural communities in the North-eastern Peruvian Amazon, to provide knowledge on the reproductive physiology of this species. The observed mean ovulation rate was 1.73 follicles, reaching a maximum diameter of 1.0 cm. After ovulation, the matured follicle luteinizes resulting in functional corpora lutea (CL). In case of oocyte fertilization, the “pregnancy” CL grow to a maximum of 2 cm in diameter, and luteal volume decreases related to the advance of pregnancy. Pregnant females have waves of follicular activity until late pregnancy, but dominant follicles do not attain the maximum diameter of pre-ovulatory follicles. Some non-ovulated follicles of 1 mm maximum diameter do not undergo atretic processes and transform to accessory CL by luteinization of the membrane granulosa, resulting in a contribution of up to 7% of the total luteal volume. All pregnant females delivered at term only 1.00 foetus, resulting in a rate of reproductive wastage of 33.3% of embryos.     

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.     

Characterization of the liver kinase B1-mouse protein-25 -Ste-20-related adaptor protein complex in adult mouse skeletal muscle

In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25α/β), and Ste-20-related adaptor protein (STRADα/β); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1(-/-)) mouse muscle. LKB1 detection in untreated control and LKB1(-/-) muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1(-/-) samples [55 ± 2.5 and 13 ± 1.5 arbitrary units (AU) in control and LKB1(-/-), respectively, P < 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 ± 5 and 8.4 ± 4 AU in control and LKB1(-/-), respectively, P < 0.01) and ion-exchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25α, and STRADβ mRNA in LKB1(-/-) muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.     

A spring-loaded release mechanism regulates domain movement and catalysis in phosphoglycerate kinase

Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement, catalysis, and efficient product release.