The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions

Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a "copper switch." This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11-21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.     

Neuronal Ca2+ signaling via caldendrin and calneurons

The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.     

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by M?ssbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.     

Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.     

Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Helices in peptoids of alpha- and beta-peptides

Peptoids of alpha- and beta-peptides (alpha- and beta-peptoids) can be obtained by shifting the amino acid side chains from the backbone carbon atoms of the monomer constituents to the peptide nitrogen atoms. They are, therefore, N-substituted poly-glycines and poly-beta-alanines, respectively. Due to the substituted nitrogen atoms, the ability for hydrogen bond formation between peptide bonds gets lost. It may be very interesting to see whether such non-natural oligomers could be regarded as foldamers, which fold into definite backbone conformers. In this paper, we provide a complete overview on helix formation in alpha- and beta-peptoids on the basis of systematic theoretical conformational analyses employing the methods of ab initio molecular orbital (MO) theory. It can be shown that the alpha- and beta-peptoid structures form helical structures with both trans and cis peptide bonds despite the missing hydrogen bonds. Obviously, the conformational properties of the backbone are more important for folding than the possibility of hydrogen bonding. There are close relationships between the helices of alpha-peptoids and poly-glycine and poly-proline helices of alpha-peptides, whereas the helices of beta-peptoids correspond to the well-known helical structures of beta-peptides as, for instance, the 3(1)-helix of beta-peptides with 14-membered hydrogen-bonded rings. Thus, alpha- and beta-peptoids enrich the field of foldamers and may be used as useful tools in peptide and protein design.     

Polymer-supported ferrocenyl oxazolines for the catalyzed highly enantioselective phenyl transfer to aldehydes

A new chiral MeO-PEG-supported ferrocenyl oxazoline was synthesized and successfully employed in the enantioselective phenyl transfer to aldehydes. The products were obtained in high yields and with excellent enantioselectivities (up to 97% ee). Furthermore, the recovery of the ferrocene was facile, and catalyst efficiency was maintained in subsequent reactions.     

The dialogue between viruses and hosts in compatible interactions

Understanding the biological principles behind virus-induced symptom expression in plants remains a longstanding challenge. By dissecting the compatible host-virus relationship temporally and genetically, we have begun to map out the relationships of its component parts. The picture that emerges is one in which host gene expression and physiology are under tight temporal control during infection.     

Neither apposition nor superposition: the compound eyes of the Chestnut Leafminer Cameraria ohridella

The two lemon-shaped compound eyes of the moth Cameraria ohridella measure in dorsal–ventral direction 263.0 μm in male and 238.9 μm in female individuals. In anterior–posterior direction no significant differences were found between the sexes, eye length being about 194.6 μm. The eyes of males consist of ca. 417 hexagonal facets, while those of females contain 367. In both sexes facet diameters are approximately 11.5 μm. Despite the size differences of the eyes in the two sexes, ultrastructurally they are identical and both possess ommatidia of 80 μm mean length. The ultrastructure of the eye is described and compared with that of other compound eyes of Lepidoptera. Anatomically the eyes represent a type intermediate between apposition and refractive superposition kind. A distal rhabdom is present in the space that in the eyes of larger moths with superposition optics is occupied by the so-called clear zone. A tracheal tapetum and longitudinal screening pigment migrations, typical of superposition but not apposition eyes are present despite the lack of a clear zone. Thus, our results support an earlier calculated minimal theoretical limit for superposition eyes.     

Inflammatory phase of bone healing initiates the regenerative healing cascade

Bone healing commences with an inflammatory reaction which initiates the regenerative healing process leading in the end to reconstitution of bone. An unbalanced immune reaction during this early bone healing phase is hypothesized to disturb the healing cascade in a way that delays bone healing and jeopardizes the successful healing outcome. The immune cell composition and expression pattern of angiogenic factors were investigated in a sheep bone osteotomy model and compared to a mechanically-induced impaired/delayed bone healing group. In the impaired/delayed healing group, significantly higher T cell percentages were present in the bone hematoma and the bone marrow adjacent to the osteotomy gap when compared to the normal healing group. This was mirrored in the higher cytotoxic T cell percentage detected under delayed bone healing conditions indicating longer pro-inflammatory processes. The highly activated periosteum adjourning the osteotomy gap showed lower expression of hematopoietic stem cell markers and angiogenic factors such as heme oxygenase and vascular endothelial growth factor. This indicates a deferred revascularization of the injured area due to ongoing pro-inflammatory processes in the delayed healing group. Results from this study suggest that there are unfavorable immune cells and factors participating in the initial healing phase. In conclusion, identifying beneficial aspects may lead to promising therapeutical approaches that might benefit further by eliminating the unfavorable factors.