Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.     

The effects of estrogen on cortisol metabolism in female baboons

We examined Cortisol (F) dynamics in female baboons $\text{(Papio anubis)}$ treated with diethylstilbestrol (DES) or estradiol (E₂) and compared values with those previously measured in nonpregnant and pregnant animals. Five regularly menstruating baboons (12–18 kg, BW) were administered 5 mg DES daily via fruit or 0.5 mg E₂/0.1ml oil sc for 30 days. Blood samples, obtained before and after treatment, were assayed for serum F concentrations and serum Cortisol binding capacity (CBC). The metabolic clearance (MCR) and production rate (PR) of F and the catabolism of i.v. administered [³H] F were examined 25 and 30 days after initiation of estrogen treatment. Compared with values in nonpregnant baboons, F metabolism in estrogen treated animals is significantly altered and is characterized by increased formation of unconjugated metabolites, decreased glucuronylation, increased excretion of unconjugated F, cortisone, and highly polar metabolites, and increased CBC. These changes induced by estrogen are similar to those observed in intact pregnant baboons and permit the suggestion that the pattern of F metabolism and the level of CBC in baboon pregnancy are the result of elevated estrogen production.However, estrogen also caused a significant decrease in the MCR and PR of F, parameters which, by contrast, are similar in intact pregnant and nonpregnant baboons. These findings indicate that while estrogen also influences the rate of F clearance and F production, these effects of estrogen are not apparent during pregnancy. Collectively, these findings allow the suggestion that estrogen is a major factor which alters F metabolism and increases serum CBC in baboon gestation. However, additional factors are operative in primate pregnancy which maintain PR and MCR of F at levels similar to those of nonpregnant baboons.     

Morphological variation and heritability in three Melanesian populations: A multivariate approach

In two previous papers Giles, Walsh and Bradley ('66) and Giles, Wyber and Walsh ('70) have shown that the inhabitants of three adjacent villages on the edge of the Markham Valley in New Guinea have significant heterogeneity in all blood group frequencies tested. The language, environment, culture, and ancestry of these people are essentially identical. The differences in the blood group frequencies were attributed to genetic drift and particularly to founder effect. In this paper the anthropometric data from these villages are analysed. Analysis of variance shows that 50% of the traits are significantly different. Multiple discriminant analysis demonstrates that the villages can be significantly separated morphologically. The heritability of each trait is determined using an analysis of within and among sibship variation. Traits with strong “genetic” components of variation and traits with strong “environmental” components contribute significantly to the morphological heterogeneity among the villages. Some speculations are presented on the cause of the “genetic” component of morphological variation.     

A New Computational Efficient Approach for Trabecular Bone Analysis using Beam Models Generated with Skeletonized Graph Technique

Micro-finite element (FE) analysis is a well established technique for the evaluation of the elastic properties of trabecular bone, but is limited in its application due to the large number of elements that it requires to represent the complex internal structure of the bone. In this paper, we present an alternative FE approach that makes use of a recently developed 3D-Line Skeleton Graph Analysis (LSGA) technique to represent the complex internal structure of trabecular bone as a network of simple straight beam elements in which the beams are assigned geometrical properties of the trabeculae that they represent. Since an enormous reduction of cputime can be obtained with this beam modeling approach, ranging from approximately 1,200 to 3,600 for the problems investigated here, we think that the FE modeling technique that we introduced could potentially constitute an interesting alternative for the evaluation of the elastic mechanical properties of trabecular bone.     

Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles

To characterize the mechanism through which myosin XI-K attaches to its principal endomembrane cargo, a yeast two-hybrid library of Arabidopsis thaliana cDNAs was screened using the myosin cargo binding domain as bait. This screen identified two previously uncharacterized transmembrane proteins (hereinafter myosin binding proteins or MyoB1/2) that share a myosin binding, conserved domain of unknown function 593 (DUF593). Additional screens revealed that MyoB1/2 also bind myosin XI-1, whereas myosin XI-I interacts with the distantly related MyoB7. The in vivo interactions of MyoB1/2 with myosin XI-K were confirmed by immunoprecipitation and colocalization analyses. In epidermal cells, the yellow fluorescent protein–tagged MyoB1/2 localize to vesicles that traffic in a myosin XI–dependent manner. Similar to myosin XI-K, MyoB1/2 accumulate in the tip-growing domain of elongating root hairs. Gene knockout analysis demonstrated that functional cooperation between myosin XI-K and MyoB proteins is required for proper plant development. Unexpectedly, the MyoB1-containing vesicles did not correspond to brefeldin A–sensitive Golgi and post-Golgi or prevacuolar compartments and did not colocalize with known exocytic or endosomal compartments. Phylogenomic analysis suggests that DUF593 emerged in primitive land plants and founded a multigene family that is conserved in all flowering plants. Collectively, these findings indicate that MyoB are membrane-anchored myosin receptors that define a distinct, plant-specific transport vesicle compartment.     

Sevoflurane modulates the activity of glyceraldehyde 3-phosphate dehydrogenase

The mechanism of inhalation anesthesia remains to be fully elucidated. While certain neuronal membrane proteins are considered sites of action, cytosolic proteins may also be targets. We hypothesize that inhaled anesthetics may act via glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which has recently been shown to participate in neuronal inhibition. We examined the effects of sevoflurane, a halogenated ether anesthetic, on the catalytic and fluorescence properties of GAPDH. Initial rates of oxidoreductase activity decreased approximately 30% at saturating levels of sevoflurane. NADH-stimulated oxidoreductase activity (25 μM NADH; 0.8 mM NAD⁺) increased with sevoflurane. Sevoflurane quenched tryptophan fluorescence emission and increased polarization. Additionally, sevoflurane increased the susceptibility of GAPDH to thermal denaturation suggesting an effect on conformation. Our findings warrant further research on sevoflurane's effect on GAPDH and indicate that this approach may lead to delineation of a novel contribution to the mechanism of anesthesia.