Interaction of hyperalgesia and sensory loss in complex regional pain syndrome type I (CRPS I)

Background

Sensory abnormalities are a key feature of Complex Regional Pain Syndrome (CRPS). In order to characterise these changes in patients suffering from acute or chronic CRPS I, we used Quantitative Sensory Testing (QST) in comparison to an age and gender matched control group.

Methods

61 patients presenting with CRPS I of the upper extremity and 56 healthy subjects were prospectively assessed using QST. The patients'' warm and cold detection thresholds (WDT; CDT), the heat and cold pain thresholds (HPT; CPT) and the occurrence of paradoxical heat sensation (PHS) were observed.

Results

In acute CRPS I, patients showed warm and cold hyperalgesia, indicated by significant changes in HPT and CPT. WDT and CDT were significantly increased as well, indicating warm and cold hypoaesthesia. In chronic CRPS, thermal hyperalgesia declined, but CDT as well as WDT further deteriorated. Solely patients with acute CRPS displayed PHS. To a minor degree, all QST changes were also present on the contralateral limb.

Conclusions

We propose three pathomechanisms of CRPS I, which follow a distinct time course: Thermal hyperalgesia, observed in acute CRPS, indicates an ongoing aseptic peripheral inflammation. Thermal hypoaesthesia, as detected in acute and chronic CRPS, signals a degeneration of A-delta and C-fibres, which further deteriorates in chronic CRPS. PHS in acute CRPS I indicates that both inflammation and degeneration are present, whilst in chronic CRPS I, the pathomechanism of degeneration dominates, signalled by the absence of PHS. The contralateral changes observed strongly suggest the involvement of the central nervous system.     

Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume

To examine the structure and function of the Na-K-Cl cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl(-) provided a ratiometric indicator of intracellular [Cl(-)]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed contemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.     

<Emphasis Type="Italic">Sphaerophragmium pulchrum</Emphasis>, a new species of rust fungi on <Emphasis Type="Italic">Albizia adinocephala</Emphasis> from Panama

Sphaerophragmium pulchrum is proposed as a new microcyclic rust species on Albizia adinocephala from Panama. Telia and spermogonia are described, illustrated, and compared to those of known species of Sphaerophragmium. Spermogonia are described for the first time for a species of Sphaerophragmium. This is the first record of this genus for the southern part of the Central American isthmus.     

Novel Type of ADP-Forming Acetyl Coenzyme A Synthetase in Hyperthermophilic Archaea: Heterologous Expression and Characterization of Isoenzymes from the Sulfate Reducer Archaeoglobus fulgidus and the Methanogen Methanococcus jannaschii

Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) right harpoon over left harpoon acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers (alpha(2), beta(2)). The coding genes for the alpha and beta subunits are located at different sites in the P. furiosus chromosome. Based on significant sequence similarity of the P. furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the alpha and beta subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus and ORF MJ0590 of M. jannaschii were cloned and functionally overexpressed in Escherichia coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P. furiosus alpha and beta subunits in an alphabeta (AF1211 and MJ0590) or betaalpha (AF1938) orientation. The data indicate that A. fulgidus and M. jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused.     

New species and new records of smut fungi from China

This paper deals with six species of smut fungi which were collected in the provinces Yunnan and Gansu in China. Among them, Anthracoidea yunnanensis and Entorrhiza guttiformis are new species. Sporisorium andropogonis-annulati, which is a new combination, Sporisorium montaniensis, Urocystis pseudoanemones, and Ustilago griffithsii are new records for China.     

Intensity of the vaccine-elicited immune response determines tumor clearance.

Tumor Ag-specific vaccines used for cancer immunotherapy can generate specific CD8 responses detectable in PBMCs and in tumor-infiltrating lymphocytes. However, human studies have shown that detection of a systemic vaccine-induced response does not necessarily correlate with the occasional instances of tumor rejection. Because this discrepancy might partially be attributable to the genetic heterogeneity of human cancers, as well as to the immunosuppressive effects of previous treatments, we turned to a mouse model in which these variables could be controlled to determine whether a relationship exists between the strength of vaccine-induced immune responses and tumor rejection. We challenged mice with the beta-galactosidase (beta-gal)-expressing tumor cells, C25.F6, vaccinated them with beta-gal-carrying viral vectors, and used quantitative RT-PCR to measure the vaccine-induced immune response of splenocytes directly ex vivo. We found that the strength of the response increased with increasing doses of beta-gal-carrying vector and/or upon boosting with a heterologous beta-gal-carrying virus. Most importantly, we found that the strength of the detected immune response against this foreign Ag strongly correlated with reduction in the number of lung metastases. The results from this mouse model have major implications for the implementation of tumor vaccines in humans.     

Molecular phylogeny and host-plant use (Lamiaceae) of the Thymogethes pollen beetles (Coleoptera)

The 24 members of the Euro-Asiatic genus Thymogethes are highly specialized pollen beetles associated as larvae with flowers of Lamiaceae Nepetoideae. All members of the genus were analysed in within the framework of an integrative taxonomy approach, which was aimed to reconstruct the phylogenetic relationships, as well as the possible pattern of evolution of their larval-host-plant association. Evidence from multiple molecular markers [COI; 16S; H3], combined with an estimation of divergence times using an average rate of 0.0177 substitutions/site/My among branches, placed the origin of the genus at a minimum of 9–10 Mya. This date of origin approximates the known evolution of the host plants in Euro-Mediterranean areas. Evidence from combined molecular and cladistic morphological analyses resulted in suitable agreement with the previously established morphology-based systematics of the genus, although members of the exilis species-group were split into three clades. The only disagreement between results of this new combined phylogeny and previous classification is in the exclusion of “Thymogethes” grenieri. This species is herein positioned outside the genus, based on molecular evidence. Our analysis depicts several Thymogethes species differentiating in the last few Mys, specifically those included in the T. lugubris species-group. Combined evidence from DNA, morphology and ancestral state parsimony reconstruction of larval-host-plant associations suggests that subtribe Menthinae likely represents the ancestral host plants, with a series of independent host shifts during the radiation of the clade, in association first with Menthinae and subsequently with Lavandulinae and Nepetinae. Steno-oligophagy is the most frequent (86%) condition, while strictly monophagous species are less numerous (14%).     

Molecular modelling of the mass density of single proteins

Using molecular dynamics (MD) simulations, the density of single proteins and its temperature dependence was modelled starting from the experimentally determined protein structure and a generic, transferable force field, without the need of prior parameterization. Although all proteins consist of the same 20 amino acids, their density in aqueous solution varies up to 10% and the thermal expansion coefficient up to twofold. To model the protein density, systematic MD simulations were carried out for 10 proteins with a broad range of densities (1.32–1.43?g/cm³) and molecular weights (7–97?kDa). The simulated densities deviated by less than 1.4% from their experimental values that were available for four proteins. Further analyses of protein density showed that it can be essentially described as a consequence of amino acid composition. For five proteins, the density was simulated at different temperatures. The simulated thermal expansion coefficients ranged between 4.3 and 7.1?×?10^?4?K^?1 and were similar to the experimentally determined values of ribonuclease-A and lysozyme (deviations of 2.4 and 14.6%, respectively). Further analyses indicated that the thermal expansion coefficient is linked to the temperature dependence of atomic fluctuations: proteins with a high thermal expansion coefficient show a low increase in flexibility at increasing temperature. A low increase in atomic fluctuations with temperature has been previously described as a possible mechanism of thermostability. Thus, a high thermal expansion coefficient might contribute to protein thermostability.     

Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY*

Transmembrane stretch M_2C from the bacterial K⁺-translocating protein KtrB is unusually long. In its middle part, termed M_2C2, it contains several small and polar amino acids. This region is flanked by the two α-helices M_2C1 and M_2C3 and may form a flexible gate at the cytoplasmic side of the membrane controlling K⁺ translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M_2C2 were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K⁺ ions, M_2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M_2B residue Asp-222R1 and M_2C3 residue Val-331R1 but not with respect to M_2C1 residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K⁺ ions, structural models of the open and closed conformations were developed.