Proton relay system in the active site of maltodextrinphosphorylase via hydrogen bonds with large proton polarizability: an FT-IR difference spectroscopy study

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4–8.4 by Fourier transform infrared (FT-IR) spectroscopy. The pK _a value of the cofactor pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely resembles the second pK _a of free PLP. FT-IR difference spectra of the binary complex of MDP+α-d-glucose-1-methylenephosphonate (Glc-1-MeP) minus native MDP were taken at pH 6.9. Following binary complex formation, two Lys residues, tentatively assigned to the active site residues Lys533 and Lys539, became deprotonated, and PLP as well as a carboxyl group, most likely of Glu637, protonated. A system of hydrogen bonds which shows large proton polarizability due to collective proton tunneling was observed connecting Lys533, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore, that this hydrogen bonded chain is highly sensitive to local electrical fields and specific interactions, respectively. In the binary complex the proton limiting structure with by far the highest probability is the one in which Glc-1-MeP is singly protonated. In a second hydrogen bonded chain the proton of Lys539 is shifted to Glu637. In the binary complex the proton remains located at Glu637. In the ternary complex composed of phosphorylase, glucose-1-phosphate (Glc-1-P), and the nonreducing end of a polysaccharide chain (primer), a second proton may be shifted to the phosphate group of Glc-1-P. In the doubly protonated phosphate group the loss of mesomeric stabilization of the phosphate ester makes the C₁–O₁ bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl carbonium ion will be a substrate for nucleophilic attack by the nonreducing terminal glucose residue of the polysaccharide chain. Received: 15 June 1997 / Revised version: 15 October 1998 / Accepted: 15 October 1998     

Phylogenetic relationships of Betula species (Betulaceae) based on nuclear ADH and chloroplast matK sequences

The phylogenetic relationships within the genus Betula (Betulaceae) were investigated using a part of the nuclear ADH gene and DNA sequences of the chloroplast matK gene with parts of its flanking regions. Two well-supported phylogenetic groups could be identified in the chloroplast DNA sequence: one containing the three American species B. lenta, B. alleghaniensis, and B. papyrifera and the other including all the other species studied. The ADH gene displayed more variation, and three main groups could be identified. In disagreement with the classical division of the genus Betula, B. schmidtii and B. nana grouped with the species in subgenus Betula, and B. ermanii grouped with species in subgenus Chamaebetula, including B. humilis and B. fruticosa. The ADH phylogeny suggests that several independent polyploidizations within the genus Betula could have taken place. The ADH and chloroplast phylogenies were in part incongruent due to the placement of B. papyrifera. The most likely reason for this seems to be cytoplasmic introgression.     

pH is decreased in transplanted rat pancreatic islets

Recent studies of transplanted pancreatic islets have indicated incomplete revascularization. We investigated the pH, in relation to oxygen tension (Po(2)), in endogenous islets and islets syngeneically transplanted to the renal subcapsular site of nondiabetic and streptozotocin-diabetic recipients. Tissue pH and Po(2) were measured using microelectrodes. In the endogenous islets, tissue pH was similar to that in arterial blood. In the transplanted islets, tissue pH was 0.11-0.15 pH units lower. No differences in islet graft pH were seen between nondiabetic and diabetic animals, and none if the islet grafts were investigated 1 day or 1 mo posttransplantation. The Po(2) in the endogenous islets was approximately 35 mmHg. Transplanted islets had a markedly lower tissue Po(2) both 1 day and 1 mo after transplantation. A negative correlation between the tissue Po(2) and the hydrogen ion concentration was seen in the 1-mo-old islet transplants in diabetic animals. In conclusion, decreased Po(2) in transplanted islets is associated with a decreased tissue pH, suggesting a shift toward more anaerobic glucose metabolism after transplantation.     

Lack of molecular genetic divergence between sea-ranched and wild sea trout (Salmo trutta)

The supportive breeding programme for sea trout (Salmo trutta) in the River Dalälven, Sweden, is based on a sea‐ranched hatchery stock of local origin that has been kept ‘closed’ to the immigration of wild genes since the late 1960s (about seven generations). In spite of an apparent potential for substantial uni directional gene flow from sea‐ranched to wild (naturally produced) trout, phenotypic differences with a presumed genetic basis have previously been observed between the two ‘stocks’. Likewise, two previous studies of allozyme and mitochondrial DNA variation based on a single year of sampling have indicated genetic differentiation. In the present study we used microsatellite and allozyme data collected over four consecutive years, and tested for the existence of overall genetic stock divergence while accounting for temporal heterogeneity. Statistical analyses of allele frequency variation (F‐statistics) and multilocus genotypes (assignment tests) revealed that wild and sea‐ranched trout were significantly different in three of four years, whereas no overall genetic divergence could be found when temporal heterogeneity among years within stocks was accounted for. On the basis of estimates of effective population size in the two stocks, and of F_ST between them, we also assessed the level of gene flow from sea‐ranched to wild trout to be ≈ 80% per generation (with a lower confidence limit of ≈ 20%). The results suggest that the reproductive success of hatchery and naturally produced trout may be quite similar in the wild, and that the genetic characteristics of the wild stock are largely determined by introgressed genes from sea‐ranched fish.     

The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices

Coiled coils are well-known as oligomerization domains, but they are also important sites of protein-protein interactions. We determined the NMR solution structure and backbone (15)N relaxation rates of a disulfide cross-linked, two-chain, 37-residue polypeptide containing the 34 C-terminal residues of striated muscle alpha-tropomyosin, TM9a(251-284). The peptide binds to the N-terminal region of TM and to the tropomyosin-binding domain of the regulatory protein, troponin T. Comparison of the NMR solution structure of TM9a(251-284) with the X-ray structure of a related peptide [Li, Y., Mui, S., Brown, J. H., Strand, J., Reshetnikova, L., Tobacman, L. S., and Cohen, C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7378-7383] reveals significant differences. In solution, residues 253-269 (like most of the tropomyosin molecule) form a canonical coiled coil. Residues 270-279, however, are parallel, linear helices, novel for tropomyosin. The packing between the parallel helices results from unusual interface residues that are atypical for coiled coils. Y267 has poor packing at the coiled-coil interface and a lower R(2) relaxation rate than neighboring residues, suggesting there is conformational flexibility around this residue. The last five residues are nonhelical and flexible. The exposed surface presented by the parallel helices, and the flexibility around Y267 and the ends, may facilitate binding to troponin T and formation of complexes with the N-terminus of tropomyosin and actin. We propose that unusual packing and flexibility are general features of coiled-coil domains in proteins that are involved in intermolecular interactions.     

Scene segmentation by spike synchronization in reciprocally connected visual areas. II. Global assemblies and synchronization on larger space and time scales

 We present further simulation results of the model of two reciprocally connected visual areas proposed in the first paper [Knoblauch and Palm (2002) Biol Cybern 87:151–167]. One area corresponds to the orientation–selective subsystem of the primary visual cortex, the other is modeled as an associative memory representing stimulus objects according to Hebbian learning. We examine the scene-segmentation capability of our model on larger time and space scales, and relate it to experimental findings. Scene segmentation is achieved by attention switching on a time-scale longer than the gamma range. We find that the time-scale can vary depending on habituation parameters in the range of tens to hundreds of milliseconds. The switching process can be related to findings concerning attention and biased competition, and we reproduce experimental poststimulus time histograms (PSTHs) of single neurons under different stimulus and attentional conditions. In a larger variant the model exhibits traveling waves of activity on both slow and fast time-scales, with properties similar to those found in experiments. An apparent weakness of our standard model is the tendency to produce anti-phase correlations for fast activity from the two areas. Increasing the inter-areal delays in our model produces alternations of in-phase and anti-phase oscillations. The experimentally observed in-phase correlations can most naturally be obtained by the involvement of both fast and slow inter-areal connections; e.g., by two axon populations corresponding to fast-conducting myelinated and slow-conducting unmyelinated axons. Received: 22 August 2001 / Accepted in revised form: 8 April 2002     

Reduced Weight Gain Due to Subclinical Anaplasma phagocytophilum (Formerly Ehrlichia phagocytophila) Infection

Tick-borne fever (TBF) is caused by the rickettsia Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) and is a common disease in sheep in areas of Norway infested by Ixodes ricinus ticks. TBF can cause both direct and indirect losses to sheep kept on tick-infested pastures. In the present work we studied a sheep flock of 26 ewes and 50 lambs on pasture from May until September. No cases of TBF had earlier been observed on this pasture. Blood samples from lambs with a reduced weekly weight gain were collected and analysed for A. phagocytophilum infection by blood smear examination. In addition, at the end of the study, sera from all lambs were analysed by an indirect fluorescent antibody assay (IFA) to determine the antibody titre to E. equi. No clinical signs of tick-borne infections were observed, except in one lamb. However, 30 (60%) of the lambs grazing on this pasture became infected with A. phagocytophilum, and the infected lambs had a reduced weight gain (mean) of 3.8 kg compared with the uninfected lambs. The present study indicates that A. phagocytophilum infection may be widespread and contribute to considerable productivity losses even on pastures with no apparent tick infestation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular phylogeny of Leptosphaeria and Phaeosphaeria

The objectives of this study were to determine the phylogenetic relationships of species of Leptosphaeria and Phaeosphaeria and evaluate the phylogenetic significance of morphological characters of the teleomorph, anamorph, and host. Sequences of the entire ITS region, including the 5.8S rDNA, of 59 isolates representing 54 species were analyzed and the phylogeny inferred using parsimony and distance analyses. Isolates grouped into three well-supported clades. The results of this study support the separation of Phaeosphaeria from Leptosphaeria sensu stricto. Leptosphaeria bicolor and the morphologically similar Leptosphaeria taiwanensis formed a separate, well-supported clade. We conclude that peridial wall morphology, anamorph characteristics, and to a lesser extent host, are phylogenetically significant at the generic level. Ascospore and conidial morphology are taxonomically useful at the species level.     

Absorption of surfactants by membranes: erythrocytes versus synthetic vesicles.

Three surfactants (chlorpromazine hydrochloride, thioridazine hydrochloride, and sodium deoxycholate) are found to absorb just as strongly into the protein-containing membranes of erythrocytes as into the phospholipid bilayers of synthetic vesicles. In the concentration region where hemolysis occurs and the Langmuir adsorption isotherm is no longer valid, one may use a phase partition model in which the erythrocyte membrane is one of the phases. The partition coefficients, expressed as the ratio of mole fraction surfactant in the membrane lipid phase to concentration of surfactant in the aqueous phase, have been calculated at the point of saturation in the erythrocyte membrane. These values are Ky = 430 M-1 (chlorpromazine, pH 5.9), 550 M-1 (deoxycholate, pH 7.6), and 640 M-1 (thioridazine, pH 5.9), in isotonic buffer at 27 degrees C. Corresponding values for synthetic vesicles made from dimyristoylphosphatidylcholine are Kx = 230 M-1 (chlorpromazine, 0.12 M buffer/KCl pH 5.9), 440 M-1 (deoxycholate, 0.20 M buffer/NaCl pH 8.0) and 510 M-1 (thioridazine, 0.12 M buffer/KCl pH 5.9), at 27 degrees C. It appears that the surfactants become an integral part of the bilayer in both vesicles and natural membranes and that the absorption is not of a peripheral nature. There is no evidence that the presence of proteins in the natural membrane inhibits the absorption of these surfactants in any way.     

Mercury and selenium distribution in human kidney cortex

Concentration of mercury and selenium were analyzed in tissue fractions of human kidney cortex samples from seven autopsy cases. Total mercury content ranged between 0.3–9.0 nmol Hg/g wet wt. Between 27–61% of the total mercury was found in the 105,000g supernatant of the tissue homogenate from six cases. In kidney cortex from the seventh case, a deceased dentist with the highest concentration of mercury, only 3% of the total mercury was found in the 105,000g supernatant and about 88% in a SDS-insoluble fraction. In this fraction the molar ratio between mercury and selenium was close to 1∶1. This study supports results from previous animal studies and indicates that mercury in human kidney cortex could be deposited in forms with different solubility. It could be of importance to speciate different forms of mercury in tissues according to solubility and association to selenium when interpretations of mercury concentrations are made.