Intermolecular interaction studies of winter flounder antifreeze protein reveal the existence of thermally accessible binding state

The physical nature underlying intermolecular interactions between two rod-like winter flounder antifreeze protein (AFP) molecules and their implication for the mechanism of antifreeze function are examined in this work using molecular dynamics simulations, augmented with free energy calculations employing a continuum solvation model. The energetics for different modes of interactions of two AFP molecules is examined in both vacuum and aqueous phases along with the water distribution in the region encapsulated by two antiparallel AFP backbones. The results show that in a vacuum two AFP molecules intrinsically attract each other in the antiparallel fashion, where their complementary charge side chains face each other directly. In the aqueous environment, this attraction is counteracted by both screening and entropic effects. Therefore, two nearly energetically degenerate states, an aggregated state and a dissociated state, result as a new aspect of intermolecular interaction in the paradigm for the mechanism of action of AFP. The relevance of these findings to the mechanism of function of freezing inhibition in the context of our work on Antarctic cod antifreeze glycoprotein (Nguyen et al., Biophysical Journal, 2002, Vol. 82, pp. 2892-2905) is discussed.     

Similarity in the fatigue behavior of trabecular bone across site and species

Within the context of improving knowledge of the structure-function relations for trabecular bone for cyclic loading, we hypothesized that the S-N curve for cyclic compressive loading of trabecular bone, after accounting for differences in monotonic strength behavior, does not depend on either site or species. Thirty-five cores of fresh-frozen elderly human vertebral trabecular bone, harvested from nine donors (mean+/-S.D., age=74+/-17 years), were biomechanically tested in compression at sigma/E(0) values (ratio of applied stress to pre-fatigue elastic modulus) ranging from 0.0026 to 0.0070, and compared against literature data (J. Biomech. Eng. 120 (1998) 647-654) for young bovine tibial trabecular bone (n=37). As reported for the bovine bone, the number of cycles to failure for the human vertebral bone was related to sigma/E(0) by a power-law relation (r(2)=0.54, n=35). Quantitative comparison of these data against those reported for the bovine bone supported our hypothesis. Namely, when the differences in mean monotonic yield strain between the two types of bone were accounted for, a single S-N curve worked well for the pooled data (r(2)=0.75, n=72). Since elderly human vertebral and young bovine tibial trabecular bone represent two very different types of trabecular bone in terms of volume fraction and architecture, these findings suggest that the dominant failure mechanisms in trabecular bone for cyclic loading occur at the ultrastructural level.     

A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection

A mouse-adapted enterovirus 71 (EV71) strain with increased virulence in mice, MP4, was generated after four serial passages of the parental EV71 strain 4643 in mice. Strain MP4 exhibited a larger plaque size, grew more rapidly, and was more cytotoxic in vitro than strain 4643. Although strains 4643 and MP4 both induced apoptosis of SK-N-SH human neuroblastoma cells, MP4 was more virulent than 4643 in 1-day-old mice (50% lethal doses, 10(2) and 10(4) PFU/mouse, respectively). Strain MP4 (5 x 10(6) PFU/mouse), but not 4643, could orally infect 7-day-old mice, resulting in rear-limb paralysis followed by death 5 to 9 days after inoculation with the virus. Histopathologically, neuronal loss and apoptosis were evident in the spinal cords as well as the brain stems of the infected mice. The limb muscles displayed massive necrosis. There was early and transient virus replication in the intestines, whereas the spinal cord, brain, and muscle became the sites of viral replication during the late phase of the infection. Virus transmission occurred among infected and noninfected cagemates, as demonstrated by the occurrence of seroconversion and the presence of viable viruses in the stool samples of the latter. Protection against EV71 challenge was demonstrated following administration of hyperimmune serum 1 day after inoculation with the virus. Nucleotide sequence analysis of the genome of EV71 strain MP4 revealed four nucleotide changes on the 5' untranslated region, three on the VP2 region, and eight on the 2C region, resulting in one and four amino acid substitutions in the VP2 and 2C proteins, respectively.     

Rapid evolution of a sexually selected trait following population establishment in a novel habitat

Colonization of novel environments creates new selection pressures. Sexually selected traits are affected by the physical and social environment and should be especially susceptible to change, but this has rarely been studied. In southern California, dark-eyed juncos, (Junco hyemalis) naturally breed in mixed-coniferous temperate forests, typically from 1500 m to 3000 m in elevation. In the early 1980s, a small population became established in a coastal habitat, the University of California, San Diego campus, which has a mild, Mediterranean climate. I show that a sexually and socially selected signaling trait--the amount of white in the tail--has declined by approximately 22% as compared to mountain juncos. I address three main factors that could explain the difference between mountain and coastal juncos: phenotypic plasticity, genetic drift, and selection. Results indicate that the first two can be ruled out as the sole cause of the plumage change, which implies that selection contributed to the genetic differentiation from the mountain population. The estimated rate of evolution is about 0.2 haldanes, comparable with rates of change in systems where individuals have been artificially introduced into new environments (e.g., guppies and Drosophila). This is the first study to demonstrate evolution of a sexually selected trait after only several generations resulting from a natural invasion into a novel environment.     

Modulation of the electron redistribution in mixed valence cytochrome C oxidase by protein conformational changes

The redistribution of two electrons in the four redox centers of cytochrome c oxidase following photodissociation of CO from the CO-bound mixed valence species has been examined by resonance Raman spectroscopy. To account for both the kinetic data, obtained from 5 micros to 2 ms, and the equilibrium results, a model is proposed in which the electron redistribution is modulated by a protein conformation transition from a nascent P(1) state to a relaxed P(2) state in a time window longer than 2 ms. In this model, all six possible two-electron reduced species are considered. The high population of species with a one-electron reduced binuclear center, in which the spectrum of heme a(3) is perturbed by the redox state of Cu(B), accounts for the significant residuals in the fitting of the kinetic data with four standard spectra derived from redox species with either zero or two electrons in the binuclear center. Under equilibrium conditions, the conformational change to the P(2) state destabilizes the redox states with only one electron in the binuclear center with respect to those with either zero or two electrons. As a result, the redox equilibrium is perturbed, and the electrons are redistributed. A simulation based on the new kinetics scheme, in which the electron redistribution is modulated by the protein conformation, gives reasonable agreement with both the equilibrium and the kinetic data, demonstrating the validity of this model.     

Stabilization of calpain large subunits by overexpression of truncated calpain small subunit in L8 myoblasts

The objectives were to investigate the function of the small subunit in the calpain system by expression of the autolytic form of this subunit in L8 myoblasts. Rat post-autolysis small subunit (21 kDa) cDNA expression plasmid was transfected into L8 myoblasts and selected by G418 containing medium. The concentrations of cytosolic micro-calpain in transfected cells, SS2 and SS3, were found to be 15.7 and 17.3% higher than that in L8Neo control cells, and the concentrations of cytosolic m-calpain in SS2 and SS3 cells were 23.3 and 16.6% higher than that in control cells (L8Neo). The half-life of micro-calpain in SS3 cells (36.5 h) was longer than that in L8Neo cells (32.4 h), while the half-life of m-calpain in SS3 cells (40.1 h) was longer than that in L8Neo cell (37.5 h). These results indicated that the expression of truncated small subunit increased the stability of micro- and m-calpain large subunits in cytosol.     

Pentagastrin-induced gastric acid secretion in the diabetic rats: role of insulin

Streptozotocin-induced diabetic rats have excessively pentagastrin-simulated acid output in which insulin seems to attenuate rather than further stimulate acid output. The aim of this study was to determine the insulin impact on pentagastrin-stimulated acid output of diabetic and non-diabetic rats to resolve whether an attenuated effect does exist. Diabetic rats were induced by the streptozotocin i.v. injection four days before acid study. Some streptozotocin-treated rats additionally received daily insulin (2.4 IU/kg) injection. Using an autotitrator, acid output was measured every five minutes by the titration of gastric perfusate. Basal output was collected for 45 min before the 90-min pentagastrin infusion (0.89 microg/kg/min). Plasma gastric inhibitory polypeptide (GIP) levels were measured. Both doses (0.067 and 0.133 IU/kg/min) of insulin infusion resulted in stimulated acid output in normal rats. The subsequent insulin infusion (0.133 IU/kg/min) for non-diabetic rats undergoing pentagastrin-treatment suppressed their stimulated acid output almost down to the basal level. Pentagastrin-stimulation led to the excessively increased acid output of diabetic rats throughout the whole infusion period (P < 0.01). Correction of hyperglycemia with insulin for diabetic rats normalized the stimulated acid output. Measured basal and stimulated plasma GIP levels of those diabetic rats during acid stimulation remained higher, regardless of insulin treatment (P < 0.05). Our results suggest that insulin has the ability to attenuate pentagastrin-stimulated acid output in rats, whereas GIP is not involved in this attenuation. This effect appears to be responsible for the excessive acid output of diabetic rats undergoing pentagastrin stimulation.