1-Methyl-4-phenylpyridine (MPP+) induces oxidative stress in the rodent

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces an irreversible parkinsonism in primates. Recent evidence suggests metabolism of MPTP to 1-methyl-4-phenylpyridine (MPP+) is required for toxicity. We have proposed that MPP+ may play a central role in the toxicity of MPTP, but direct assessment of the effects of MPP+ in brain is difficult. Therefore, we have sought to define the mechanism of peripheral MPP+ toxicity in the rat and mouse. Systemically administered MPP+ produced its major pathology in the lung and was typified by perivascular edema. An increase in plasma glutathione disulfide concentrations also resulted, suggesting that MPP+ in analogy to paraquat produces oxidative stress. In addition, the lethality of MPP+ in the mouse was increased by dietary selenium deficiency. These results define in both pathological and chemical terms the potent systemic toxicity of MPP+ and suggest that MPP+, because of its high concentration in primate brain, has the potential to play an important role in the CNS toxicity of MPTP.     

Towards an interpretation of 13C chemical shifts in bathorhodopsin,a functional intermediate of a G-protein coupled receptor

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired ¹³C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the ¹³C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the ¹³C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.     

Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.     

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.     

Maize ears sculptured in 12th and 13th centuryA.D. India as indicators of pre-columbian diffusion

Evidence for the presence of maize (Lea. mays, Poaceae) in India prior to traditional European contact is found in stone sculptures of maize ears in the 12th and 13th century (and earlier) Hoysala temples in southern India near Mysore. These “ears” present the morphology of maize in such intricate and specifically variable representations that it would have been impossible for sculptors to have imagined the variability consistently and realistically without large numbers of actual maize ears as models. No other natural model could supply this variability. We should search for other crops and cultural artifacts that would have diffused with maize across the oceans before 1492 A.D.     

Successful Triple Immuno - Enzymatic Method Employing Primary Antibodies from Same Species and Same Immunoglobulin Subclass

Protocols for immunohistochemical (IHC) detection of multiple antigens in the same tissue sections have been developed using primary antibodies directly conjugated to different enzymes or fluorochromes, or ones that have been raised in different species, or from different immunoglobulin (Ig) classes or subclasses. For antibodies lacking such dissimilarities, very few proposals have been published with varying degrees of generalizability. In this report we present a successful triple IHC protocol engaging three unconjugated monoclonal primary antibodies raised in the same species and of the same Ig subclass. Compared to other methods, our results showed that denaturation of the preceding reaction complex by microwave heating, combined with additional suppression of enzyme activity, enabled the detection of all three reactions by using the same detection system, with no cross reaction observed. Moreover, expression patterns of each of the three antigens in the triple stained sections, was found to be similar to the pattern observed when single staining was performed. Unlike previous reports, no damage of targeted antigens or tissues did occur following this protocol. Furthermore, the contrast of the colors employed was investigated by computerized color deconvolution, and the three reactions products were successfully separated into three individual images that could be used for further objective quantification.Key words: triple immunohistochemistry, immunoenzyme, same species antibodies     

Effect of fiber orientation and strain rate on the nonlinear uniaxial tensile material properties of tendon

Tendons are exposed to complex loading scenarios that can only be quantified by mathematical models, requiring a full knowledge of tendon mechanical properties. This study measured the anisotropic, nonlinear, elastic material properties of tendon. Previous studies have primarily used constant strain-rate tensile tests to determine elastic modulus in the fiber direction. Data for Poisson's ratio aligned with the fiber direction and all material properties transverse to the fiber direction are sparse. Additionally, it is not known whether quasi-static constant strain-rate tests represent equilibrium elastic tissue behavior. Incremental stress-relaxation and constant strain-rate tensile tests were performed on sheep flexor tendon samples aligned with the tendon fiber direction or transverse to the fiber direction to determine the anisotropic properties of toe-region modulus (E0), linear-region modulus (E), and Poisson's ratio (v). Among the modulus values calculated, only fiber-aligned linear-region modulus (E1) was found to be strain-rate dependent. The E1 calculated from the constant strain-rate tests were significantly greater than the value calculated from incremental stress-relaxation testing. Fiber-aligned toe-region modulus (E(1)0 = 10.5 +/- 4.7 MPa) and linear-region modulus (E1 = 34.0 +/- 15.5 MPa) were consistently 2 orders of magnitude greater than transverse moduli (E(2)0 = 0.055 +/- 0.044 MPa, E2 = 0.157 +/- 0.154 MPa). Poisson's ratio values were not found to be rate-dependent in either the fiber-aligned (v12 = 2.98 +/- 2.59, n = 24) or transverse (v21 = 0.488 +/- 0.653, n = 22) directions, and average Poisson's ratio values in the fiber-aligned direction were six times greater than in the transverse direction. The lack of strain-rate dependence of transverse properties demonstrates that slow constant strain-rate tests represent elastic properties in the transverse direction. However, the strain-rate dependence demonstrated by the fiber-aligned linear-region modulus suggests that incremental stress-relaxation tests are necessary to determine the equilibrium elastic properties of tendon, and may be more appropriate for determining the properties to be used in elastic mathematical models.