Determination of Ligand Pathways in Globins: APOLAR TUNNELS VERSUS POLAR GATES*

Although molecular dynamics simulations suggest multiple interior pathways for O₂ entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.     

Triplet-state conformational changes in 15-cis-spheroidene bound to the reaction center from Rhodobacter sphaeroides 2.4.1 as revealed by time-resolved EPR spectroscopy: strengthened hypothetical mechanism of triplet-energy dissipation

Time-resolved EPR spectra of 15-cis-spheroidene bound to the reaction center from Rhodobacter sphaeroides 2.4.1 were recorded at low temperatures. (1) A three-component analysis of the spectral-data matrices by singular-value decomposition followed by global fitting identified the transformation of the triplet carotenoid, (3)Car(I) --> (3)Car(II); during this process, the leak of the triplet population was suggested. A four-component analysis suggested the presence of a representative intermediate, (3)Car(R), that forms a leak channel of the triplet population. (2) A theoretical calculation of the zero-field splitting parameters, |D| and |E|, by the use of a polyene model, showed that the transformation, (3)Car(I) --> (3)Car(R) --> (3)Car(II), accompanies the conformational changes of (0 degrees , 0 degrees , 0 degrees ) --> (+20 degrees , -20 degrees , +20 degrees ) --> (+45 degrees , -40 degrees , +40 degrees ) around the central cis C15=C15', trans C13=C14, and trans C11=C12 bonds, respectively. (3) The initial, rapid decrease followed by the inversion of spin polarization along the z axis of (3)Car was observed, which was correlated with a change in the spin angular momentum. (4) In reference to the binding pocket of the Car, determined by X-ray crystallography, the conformational changes were ascribed to the intrinsic isomerization property of 15-cis (3)Car as well as the Car-peptide intermolecular interaction; a detailed picture was proposed. All of the above results support the mechanism of triplet-energy dissipation proposed previously: the rotational motions around the central double bonds cause a change in the orbital angular momentum and, through the spin-orbit coupling, a change in the spin angular momentum, which enhances the T(1) --> S(0) intersystem crossing dissipating the triplet energy.     

Integrin-regulated secretion of interleukin 4: A novel pathway of mechanotransduction in human articular chondrocytes

Chondrocyte function is regulated partly by mechanical stimulation. Optimal mechanical stimulation maintains articular cartilage integrity, whereas abnormal mechanical stimulation results in development and progression of osteoarthritis (OA). The responses of signal transduction pathways in human articular chondrocytes (HAC) to mechanical stimuli remain unclear. Previous work has shown the involvement of integrins and integrin-associated signaling pathways in activation of plasma membrane apamin-sensitive Ca2+-activated K+ channels that results in membrane hyperpolarization of HAC after 0. 33 Hz cyclical mechanical stimulation. To further investigate mechanotransduction pathways in HAC and show that the hyperpolarization response to mechanical stimulation is a result of an integrin-dependent release of a transferable secreted factor, we used this response. Neutralizing antibodies to interleukin 4 (IL-4) and IL-4 receptor alpha inhibit mechanically induced membrane hyperpolarization and anti-IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1beta (IL-1beta) and cytokine receptors, interleukin 1 receptor type I and the common gamma chain/CD132 (gamma) have no effect on me- chanically induced membrane hyperpolarization. Chondrocytes from IL-4 knockout mice fail to show a membrane hyperpolarization response to cyclical mechanical stimulation. Mechanically induced release of the chondroprotective cytokine IL-4 from HAC with subsequent autocrine/paracrine activity is likely to be an important regulatory pathway in the maintenance of articular cartilage structure and function. Finally, dysfunction of this pathway may be implicated in OA.     

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptor (AMPAR) endocytosis is essential for N-methyl-D-aspartate-induced neuronal apoptosis

Excessive activation of the N-methyl-d-aspartate subtype glutamate receptor (NMDAR) is thought to be involved in mediating programmed cell death (apoptosis) in numerous central nervous diseases. However, the underlying mechanisms remain unknown. We report here that stimulation of NMDARs activates intracellular signaling cascades leading to apoptosis and facilitates clathrin-dependent endocytosis of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors (AMPARs). Both broad spectrum inhibitors of clathrin-dependent endocytotic processes and a specific inhibitor of AMPAR endocytosis selectively inhibit NMDA-induced apoptosis without affecting apoptosis produced by staurosporine. These results demonstrate that clathrin-dependent endocytosis of AMPARs is an essential step in NMDAR-mediated neuronal apoptosis. Our study not only identifies a previously unsuspected step in NMDA-induced apoptosis but also demonstrates that AMPAR endocytosis, in addition to attenuating synaptic strength as previously demonstrated in models of synaptic plasticity, may play a critical role in mediating other important intracellular pathways.     

Differential suppression of hyperglycemic,feeding, and neuroendocrine responses in anorexia

We have used the anorexia shown by rats given hypertonic saline to drink to investigate central mechanisms that can inhibit feeding. Rats dehydrated in this manner for 3 or 5 days showed a severe attenuation of the compensatory feeding observed after an overnight fast compared with control euhydrated rats or rats whose food was restricted to match the intake of anorexic rats. Food intake after injections of 2-deoxy-d-glucose (2-DG) was also significantly decreased in dehydrated animals compared with that after a 2-DG injection given before dehydration. However, all the dehydrated animals demonstrated a robust eating response after water was returned whether they had received injection of 2-DG or vehicle. Despite a profound reduction in 2-DG-induced feeding, other glucoregulatory responses to 2-DG remained intact in dehydrated animals. After 2-DG injection, corticosterone secretion and blood glucose were significantly elevated from preinjection values whether or not animals were dehydrated. Thus the mechanisms responsible for anorexia in dehydrated animals specifically target stimulatory feeding pathways but leave intact other counterregulatory glucometabolic motor events.     

Variation among natural isolates of Neurospora on small spatial scales

Although species of Neurospora are among the most studied model organisms in genetics and biochemistry, basic questions remain with respect to their ecology and population biology. In this study, we sought to clarify relationships among individuals over a small spatial scale, toward assessing both local variation and mode of colonization. Isolates of Neurospora were collected after fires in the Florida Everglades (May 1999), where abundant colonies appeared on diverse plants, including grasses and woody shrubs. Colonies were sampled in a linear fashion from two adjacent scorched sugarcane stems at one site and from a burned woody shrub at a distant second site. Species and mating types were assigned based on crossing behavior. Variation at two loci, het-c and frq, was determined by direct sequencing of PCR products. The results demonstrated substantial within- and among-species variation on a small scale, with up to three species and six different haplotypes occurring on a single stem. In total, four species and more than 10 genetically distinct individuals (haplotypes) were present across the three stems, often with multiple individuals occupying the same position. A permutation analysis revealed that individuals were not distributed randomly and that adjacent nodes on cane stems were more likely than chance to be colonized by the same haplotype. This suggests that visible eruptions of conidia on burned plants reflect substantial vegetative mycelial spread through subsurface tissues after primary colonization. Results also revealed that adjacent isolates from a single plant can possess different functional alleles at het-c, an observation meaningful in the context of the proposed role of het-c in self recognition.