Dopamine-independent locomotor actions of amphetamines in a novel acute mouse model of Parkinson disease

Brain dopamine is critically involved in movement control, and its deficiency is the primary cause of motor symptoms in Parkinson disease. Here we report development of an animal model of acute severe dopamine deficiency by using mice lacking the dopamine transporter. In the absence of transporter-mediated recycling mechanisms, dopamine levels become entirely dependent on de novo synthesis. Acute pharmacological inhibition of dopamine synthesis in these mice induces transient elimination of striatal dopamine accompanied by the development of a striking behavioral phenotype manifested as severe akinesia, rigidity, tremor, and ptosis. This phenotype can be reversed by administration of the dopamine precursor, L-DOPA, or by nonselective dopamine agonists. Surprisingly, several amphetamine derivatives were also effective in reversing these behavioral abnormalities in a dopamine-independent manner. Identification of dopamine transporter- and dopamine-independent locomotor actions of amphetamines suggests a novel paradigm in the search for prospective anti-Parkinsonian drugs.     

Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases

Serum amyloid A protein (SAA) is an acute-phase reactant, known to mediate pro-inflammatory cellular responses. This study reports that CLA-1 (CD36 and LIMPII Analogous-1; human orthologue of the Scavenger Receptor Class B Type I (SR-BI)) mediates SAA uptake and downstream SAA signaling. Flow cytometry experiments revealed more than a 5-fold increase of Alexa-488 SAA uptake in HeLa cells stably transfected with CLA-1. Alexa 488-HDL uptake directly correlated with SAA uptake when determined in several CLA-1 stably transfected HeLa cell clones expressing various levels of CLA-1. SAA directly binds to CLA-1 as determined by cross-linking and colocalization of anti-CLA-1 antibody with SAA. SAA was co-internalized with transferrin to the endocytic recycling compartment pointing to a potential site of SAA metabolism. Alexa-488 SAA uptake in the CLA-1-overexpressing HeLa cells, as well as in THP-1 monocyte cell line, can be efficiently blocked by unlabeled SAA, high density lipoprotein, and other CLA-1 ligands. At the same time, markedly enhanced levels of phosphorylation of the mitogen-activated protein kinases (MAPKs), ERK1/2, and p38, were observed in cells stably transfected with CLA-1 cells following SAA stimulation when compared with mock transfected cells. The levels of the SAA-induced interleukin-8 (IL-8) secretion by CLA-1-overexpressing cells also significantly exceeded (5- to 10-fold) those detected for control cells. Synthetic amphipathic peptides possessing a structural alpha-helical motif inhibited SAA-induced activation of both MAPKs and IL-8 secretion in THP-1 cells. The results of this study demonstrate for the first time that CLA-1 functions as an endocytic SAA receptor and is involved in SAA-mediated cell signaling events associated with the immune-related and inflammatory effects of SAA.     

Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.     

Backbone and side chain dynamics of mutant calmodulin-peptide complexes

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.     

Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae

Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae. From an infected juvenile of an unidentified plectid species the 16S rRNA gene sequence of Pasteuria sp. was obtained. Substantial sequence divergence from described Pasteuria species and its phylogenetic position on molecular trees indicate that this Pasteuria sp. could be considered as a new species. Preliminary results of the analysis of DNA phylogeny of Pasteuria spp. and their nematode hosts provide evidence for incongruence of their phylogenetic history and of host switching events during evolution of the bacterial parasites.     

The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.     

Functional characterization of a small conductance GIRK channel in rat atrial cells

Muscarinic K+ (KACh) channels are key determinants of the inhibitory synaptic transmission in the heart. These channels are heterotetramers consisting of two homologous subunits, G-protein-gated inwardly rectifying K+ (GIRK)1 and GIRK4, and have unitary conductance of approximately 35 pS with symmetrical 150 mM KCl solutions. Activation of atrial KACh channels, however, is often accompanied by the appearance of openings with a lower conductance, suggesting a functional heterogeneity of G-protein-sensitive ion channels in the heart. Here we report the characterization of a small conductance GIRK (scGIRK) channel present in rat atria. This channel is directly activated by Gbetagamma subunits and has a unitary conductance of 16 pS. The scGIRK and KACh channels display similar affinities for Gbetagamma binding and are frequently found in the same membrane patches. Furthermore, Gbetagamma-activated scGIRK channels--like their KACh counterparts--exhibit complex gating behavior, fluctuating among four functional modes conferred by the apparent binding of a different number of Gbetagamma subunits to the channel. The electrogenic efficacy of the scGIRK channels, however, is negligible compared to that of KACh channels. Thus, Gbetagamma subunits employ the same signaling strategy to regulate two ion channels that are apparently endowed with very different functions in the atrial membrane.     

Energy-dependent transformation of F0.F1-ATPase in Paracoccus denitrificans plasma membranes

F(0).F(1)-ATP synthase in tightly coupled inside-out vesicles derived from Paracoccus denitrificans catalyzes rapid respiration-supported ATP synthesis, whereas their ATPase activity is very low. In the present study, the conditions required to reveal the Deltamu(H+)-generating ATP hydrolase activity of the bacterial enzyme have been elucidated. Energization of the membranes by respiration results in strong activation of the venturicidin-sensitive ATP hydrolysis, which is coupled with generation of Deltam?(H+). Partial uncoupling stimulates the proton-translocating ATP hydrolysis, whereas complete uncoupling results in inhibition of the ATPase activity. The presence of inorganic phosphate is indispensable for the steady-state turnover of the Deltam?(H+)-activated ATPase. The collapse of Deltam?(H+) brings about rapid deactivation of the enzyme, which has been subjected to pre-energization. The rate and extent of the deactivation depend on protein concentration, i.e. the more vesicles are present in the assay mixture, the higher the rate and extent of the deactivation is seen. Sulfite and the ADP-trapping system protect ATPase against the Deltam?(H+) collapse-induced deactivation, whereas phosphate delays the rate of deactivation. A low concentration of ADP (<1 microm) increases the rate of deactivation. Taken together, the results suggest that latent proton-translocating ATPase in P. denitrificans is kinetically equivalent to the previously characterized ADP(Mg2+)-inhibited, azide-trapped bovine heart mitochondrial F(0).F(1)-ATPase (Galkin, M. A., and Vinogradov, A. D. (1999) FEBS Lett. 448, 123-126). A Deltam?(H+)-sensitive mechanism operates in P. denitrificans that prevents physiologically wasteful consumption of ATP by F(0).F(1)-ATPase (synthase) complex when the latter is unable to maintain certain value of Deltam?(H+).     

The genetic or mythical ancestry of descent groups: lessons from the Y chromosome

Traditional societies are often organized into descent groups called "lineages," "clans," and "tribes." Each of these descent groups claims to have a common ancestor, and this ancestry distinguishes the group's members from the rest of the population. To test the hypothesis of common ancestry within these groups, we compared ethnological and genetic data from five Central Asian populations. We show that, although people from the same lineage and clan share generally a recent common ancestor, no such common ancestry is observed at the tribal level. Thus, a tribe might be a conglomerate of clans who subsequently invented a mythical ancestor to strengthen group unity.