Copper binding to the Alzheimer’s disease amyloid precursor protein

Alzheimer’s disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called Aβ by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce Aβ levels and hence a molecular understanding of the interaction between metal and protein could lead to the development of novel therapeutics to treat the disease. We have recently determined the three-dimensional structures of apo and copper bound forms of CuBD. The structures provide a mechanism by which CuBD could readily transfer copper ions to other proteins. Importantly, the lack of significant conformational changes to CuBD on copper binding suggests a model in which copper binding affects the dimerisation state of APP leading to reduction in Aβ production. We thus predict that disruption of APP dimers may be a novel therapeutic approach to treat Alzheimer’s disease. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.     

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H₂O₂) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3β/GSK-3β ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H₂O₂ (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3β phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H₂O₂ levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.     

High rates of net ecosystem carbon assimilation by Brachiara pasture in the Brazilian Cerrado

To investigate the consequences of land use on carbon and energy exchanges between the ecosystem and atmosphere, we measured CO₂ and water vapour fluxes over an introduced Brachiara brizantha pasture located in the Cerrado region of Central Brazil. Measurements using eddy covariance technique were carried out in field campaigns during the wet and dry seasons. Midday CO₂ net ecosystem exchange rates during the wet season were ?40 μmol m^?2 s^?1, which is more than twice the rate found in the dry season (?15 μmol m^?2 s^?1). This was observed despite similar magnitudes of irradiance, air and soil temperatures. During the wet season, inferred rates of canopy photosynthesis did not show any tendency to saturate at high solar radiation levels, with rates of around 50 μmol m^?2 s^?1 being observed at the maximum incoming photon flux densities of 2200 μmol m^?2 s^?1. This contrasted strongly to the dry period when light saturation occurred with 1500 μmol m^?2 s^?1 and with maximum canopy photosynthetic rates of only 20 μmol m^?2 s^?1. Both canopy photosynthetic rates and night‐time ecosystem CO₂ efflux rates were much greater than has been observed for cerrado native vegetation in both the wet and dry seasons. Indeed, observed CO₂ exchange rates were also much greater than has previously been reported for C₄ pastures in the tropics. The high rates in the wet season may have been attributable, at least in part, to the pasture not being grazed. Higher than expected net rates of carbon acquisition during the dry season may also have been attributable to some early rain events. Nevertheless, the present study demonstrates that well‐managed, productive tropical pastures can attain ecosystem gas exchange rates equivalent to fertilized C₄ crops growing in the temperate zone.     

Leptins and leptin receptor expression in the goldfish (Carassius auratus). Regulation by food intake and fasting/overfeeding conditions

Intermedin (IMD)(1-53) is a novel member of the calcitonin gene-related peptide superfamily and has potent cardioprotective effects against myocardial injury induced by ischemia-reperfusion (I/R). To explore the mechanism of the IMD(1-53) cardioprotective effect, we studied the anti-oxidant effects of IMD(1-53) on myocardial injury induced by I/R in vivo in rat and H(2)O(2) treatment in vitro in rat cardiomyocytes. Compared with sham treatment, I/R treatment induced severe lipid peroxidation injury in rat myocardium: plasma malondialdehyde (MDA) content and myocardial LDH activity was increased by 34% and 85% (all P<0.01); Mn-superoxide dismutase (Mn-SOD) and catalase (CAT) activity was reduced 80% and 86% (all P<0.01), respectively, and the protein levels of the NADPH oxidase complex subunits gp91(phox) and p47(phox) were markedly increased, by 86% (P<0.05) and 95% (P<0.01), respectively; IMD(1-53) treatment ameliorated lipid peroxidation injury: plasma MDA content and myocardial LDH activity was decreased by 30% (P<0.05) and 36% (P<0.01); Mn-SOD and CAT activity was elevated 1.0- and 4.3-fold (all P<0.01), respectively; and the protein levels of gp91(phox) and p47(phox) were reduced, by 28% and 36% (both P<0.05), respectively. Concurrently, IMD(1-53) treatment markedly promoted cell viability and inhibited apoptosis in cardiomyocytes as compared with H(2)O(2) treatment alone. Furthermore, IMD(1-53) increased the ratio of p-ERK to ERK by 66% (P<0.05) as compared with I/R alone, and the protective effect of IMD(1-53) on H(2)O(2)-induced apoptosis was abolished by preincubation with PD98059, a MEK inhibitor. IMD(1-53) may improve the oxidative stress injury induced by I/R via inhibiting the production of reactive oxygen species and enhancing ERK phosphorylation.     

Life-long consequences of postnatal normoxia exposure in rats raised at high altitude

We tested the hypothesis that exposure of high-altitude (HA) rats to a period of postnatal normoxia has long-term consequences on the ventilatory and hematological acclimatization in adults. Male and female HA rats (3,600 m, Po(2) ? 100 Torr; La Paz, Bolivia) were exposed to normal room air [HA control (HACont)] or enriched oxygen (32% O(2); Po(2) ? 160 Torr) from 1 day before to 15 days after birth [HA postnatal normoxia (HApNorm)]. Hematocrit and hemoglobin values were assessed at 2, 12, and 32 wk of age. Cardiac and lung morphology were assessed at 12 wk by measuring right ventricular hypertrophy (pulmonary hypertension index) and lung air space-to-tissue ratio (indicative of alveolarization). Respiratory parameters under baseline conditions and in response to 32% O(2) for 10 min (relieving the ambient hypoxic stimulus) were measured by whole body plethysmography at 12 wk. Finally, we performed a survival analysis up to 600 days of age. Compared with HACont, HApNorm rats had reduced hematocrit and hemoglobin levels at all ages (both sexes); reduced right ventricular hypertrophy (both sexes); lower air space-to-tissue ratio in the lungs (males only); reduced CO(2) production rate, but higher oxygen uptake (males only); and similar respiratory frequency, tidal volume, and minute ventilation. When breathing 32% O(2), HApNorm male rats had a stronger decrease of minute ventilation than HACont. HApNorm rats had a marked tendency toward longer survival throughout the study. We conclude that exposure to ambient hypoxia during postnatal development in HA rats has deleterious consequences on acclimatization to hypoxia as adults.     

Physiological and biochemical features of embryogenic and non-embryogenic peach palm (<Emphasis Type="Italic">Bactris gasipaes</Emphasis> Kunth) cultures

Both embryogenic and non-embryogenic peach palm (Bactris gasipaes Kunth) cultures arise during somatic embryogenesis induction, and both tissue types are often observed growing side-by-side from the same explant. To better understand why this occurs, samples from each tissue type were analyzed for their endogenous concentrations of indole-3-acetic acid (IAA), abscisic acid (ABA), polyamines, and amino acids with high-performance liquid chromatography and for total phenolics with spectrophotometry. Embryogenic cultures contained significantly higher concentrations of IAA, ABA, and total amino acids, whereas non-embryogenic tissue contained more total polyamines and phenolics. The greater IAA concentrations in embryogenic cultures supported the role of that hormone as a marker of embryogenic potential. Putrescine was especially prevalent in non-embryogenic cultures; however, the decreased putrescine/spermine + spermidine ratio in embryogenic cultures added support to the conclusions of previous studies in other species that this can serve as a marker of embryogenic competence. Though embryogenic cultures contained higher total amino acids, each culture type had different concentrations of specific amino acids.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.