Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.     

Effects of afforestation on water yield: a global synthesis with implications for policy

Carbon sequestration programs, including afforestation and reforestation, are gaining attention globally and will alter many ecosystem processes, including water yield. Some previous analyses have addressed deforestation and water yield, while the effects of afforestation on water yield have been considered for some regions. However, to our knowledge no systematic global analysis of the effects of afforestation on water yield has been undertaken. To assess and predict these effects globally, we analyzed 26 catchment data sets with 504 observations, including annual runoff and low flow. We examined changes in the context of several variables, including original vegetation type, plantation species, plantation age, and mean annual precipitation (MAP). All of these variables should be useful for understanding and modeling the effects of afforestation on water yield. We found that annual runoff was reduced on average by 44% (±3%) and 31% (±2%) when grasslands and shrublands were afforested, respectively. Eucalypts had a larger impact than other tree species in afforested grasslands (P=0.002), reducing runoff (90) by 75% (±10%), compared with a 40% (±3%) average decrease with pines. Runoff losses increased significantly with plantation age for at least 20 years after planting, whether expressed as absolute changes (mm) or as a proportion of predicted runoff (%) (P<0.001). For grasslands, absolute reductions in annual runoff were greatest at wetter sites, but proportional reductions were significantly larger in drier sites (P<0.01 and P<0.001, respectively). Afforestation effects on low flow were similar to those on total annual flow, but proportional reductions were even larger for low flow (P<0.001). These results clearly demonstrate that reductions in runoff can be expected following afforestation of grasslands and shrublands and may be most severe in drier regions. Our results suggest that, in a region where natural runoff is less than 10% of MAP, afforestation should result in a complete loss of runoff; where natural runoff is 30% of precipitation, it will likely be cut by half or more when trees are planted. The possibility that afforestation could cause or intensify water shortages in many locations is a tradeoff that should be explicitly addressed in carbon sequestration programs.     

Muscle mechanical advantage of human walking and running: implications for energy cost.

Muscular forces generated during locomotion depend on an animal's speed, gait, and size and underlie the energy demand to power locomotion. Changes in limb posture affect muscle forces by altering the mechanical advantage of the ground reaction force (R) and therefore the effective mechanical advantage (EMA = r/R, where r is the muscle mechanical advantage) for muscle force production. We used inverse dynamics based on force plate and kinematic recordings of humans as they walked and ran at steady speeds to examine how changes in muscle EMA affect muscle force-generating requirements at these gaits. We found a 68% decrease in knee extensor EMA when humans changed gait from a walk to a run compared with an 18% increase in hip extensor EMA and a 23% increase in ankle extensor EMA. Whereas the knee joint was extended (154-176 degrees) during much of the support phase of walking, its flexed position (134-164 degrees) during running resulted in a 5.2-fold increase in quadriceps impulse (time-integrated force during stance) needed to support body weight on the ground. This increase was associated with a 4.9-fold increase in the ground reaction force moment about the knee. In contrast, extensor impulse decreased 37% (P < 0.05) at the hip and did not change at the ankle when subjects switched from a walk to a run. We conclude that the decrease in limb mechanical advantage (mean limb extensor EMA) and increase in knee extensor impulse during running likely contribute to the higher metabolic cost of transport in running than in walking. The low mechanical advantage in running humans may also explain previous observations of a greater metabolic cost of transport for running humans compared with trotting and galloping quadrupeds of similar size.     

Origin and ultrastructure of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum

A cell observation chamber was designed to perform continuous photomicroscopic observations of hyphal anastomosis and the origin of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum. These data were correlated with ultrastructural features of intra-hyphal hyphae. Hyphal fusions occurred commonly in either species of Trichophyton when incubated alone. In T. terrestre, empty phyphal segments adjoined by live units were invaded at the septa from both directions by new hyphal ingrowth. Continuous observations revealed that the intra-hyphal hyphae subsequently anastomosed via a lateral fusion peg. Similar intra-hyphal hyphae were shown in T. rubrum. Electron microscopic studies revealed ascomycetous septa in both conventional hyphae and intra-hyphal hyphae. For the latter, the cytoplasm and wall of the inner hypha were bounded by cytoplasmic organelles and another cell wall of the outer hypha.     

The intravenous distribution of a radiolabeled, potentially useful cluster ion of copper and penicillamine

The 64Cu-radiolabeled mixed valence cluster ion of copper and penicillamine (Cu(I)8Cu(II)6Pen12Cl5-) is easily prepared and purified in a 30 minute procedure. Mice were dosed intravenously with this substance at 0.1 mg/kg, and the distribution of the isotope among organs and tissues was quantified by gamma scintillometry of the isotope's positron annihilation radiation. The largest concentration was found in urine (a 96-fold increase over whole body specific activity at 20 minutes), and this finding is consistent with the compound's inulin-like renal clearance. Isotope levels were also elevated in kidney and liver tissues (2.36-and 3.62-fold, respectively), while all other organs and tissues examined were found to be depleted of the label. A pre-injection of unlabeled cluster at 60 mg/kg effectively blocked radiolabel interaction with liver tissue. This intensely red-violet compound did not stain viable liver cells, and it was not significantly decomposed by liver homogenates over a three hour period. These results suggest cluster interaction at saturable binding sites on liver cell surfaces. The unlabeled cluster shows no indication of toxicity, and the labeled version might have biomedical applications.     

Pirenzepine block of ACH-induced mucus secretion in tracheal submucosal gland cells

Muscarinic stimulation of mucus secretion, as measured by the release of [3H]glycoprotein, was studied in explants from the tracheal epithelium of weanling swine. The mucus glycoprotein secretion was transient, ceasing within the first 10 min of a continuous exposure to 100 microM ACh. Increasing the solution's osmotic pressure did not alter basal mucus glycoprotein secretion. Mucus glycoprotein secretion was inhibited by 2-10 microM PZP, indicating that the M3 muscarinic receptors mediate cholinergic stimulation of mucus production.