Klaus Eschrich

Plastic Neuronal Remodeling Is Impaired in Patients with Alzheimer’s Disease Carrying Apolipoprotein ε4 Allele

A relationship between the apolipoprotein E (apoE) genotype and the risk to develop Alzheimer’s disease has been established recently. Apolipoprotein synthesis is implicated in developmental processes and in neuronal repair of the adult nervous system. In the present study, we investigated the influence of the apolipoprotein polymorphism on the severity of neuronal degeneration and the extent of plastic dendritic remodeling in Alzheimer’s disease. Changes in length and arborization of dendrites of Golgi-impregnated neurons in the basal nucleus of Meynert, locus coeruleus, raphe magnus nucleus, medial amygdaloid nucleus, pedunculopontine tegmental nucleus, and substantia nigra were analyzed after three-dimensional reconstruction. Patients with either one or two apoE ε4 alleles not only showed a more severe degeneration in all areas investigated than in patients lacking the apoE 4 allele but also revealed significantly less plastic dendritic changes. ApoE ε4 allele copy number, furthermore, had a significant effect on the pattern of dendritic arborization. Moreover, the relationship between the intensity of dendritic growth and both the extent of neuronal degeneration and the stage of the disease seen in patients lacking the apoE ε4 allele was very weak in the presence of one ε4 allele and completely lost in patients homozygous for the ε4 allele. The results provide direct evidence that neuronal reorganization is affected severely in patients with Alzheimer’s disease carrying the apoE ε4 allele. This impairment of neuronal repair might lead to a more rapid functional decompensation, thereby contributing to an earlier onset and more rapid progression of the disease.

Rapid Identification of Viridans Streptococci by Mass Spectrometric Discrimination

ABSTRACT Viridans streptococci (VS) are responsible for several systemic diseases, such as endocarditis, abscesses, and septicemia. Unfortunately, species identification by conventional methods seems to be more difficult than species identification of other groups of bacteria. The aim of the present study was to evaluate the use of cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the rapid identification of 10 different species of VS. A total of 99 VS clinical isolates, 10 reference strains, and 20 strains from our in-house culture collection were analyzed by MALDI-TOF-MS. To evaluate the mass-spectrometric discrimination results, all strains were identified in parallel by phenotypic and genotypic methods. MALDI-TOF-MS identified 71 isolates as the mitis group, 23 as the anginosus group, and 5 as Streptococcus salivarius. Comparison of the species identification results obtained by the MALDI-TOF-MS analyses and with the phenotypic/genotypic identification systems showed 100% consistency at the species level. Thus, MALDI-TOF-MS seems to be a rapid and reliable method for the identification of species of VS from clinical samples.

Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease

Summary. The activities of hexokinase, aldolase, pyruvate kinase, lactate dehydrogenase and glucose 6-phosphate dehydrogenase were determined in brains of patients with Alzheimers disease (AD) and in age matched controls. For pyruvate kinase and lactate dehydrogenase a significant increase in specific activity was found in frontal and temporal cortex of AD brains, while the activities of aldolase and hexokinase are not changed. Glucose 6-phosphate dehydrogenase activity was significantly reduced in hippocampus. The increase of some glycolytic enzyme activities is correlated with increased contents of lactate dehydrogenase and glial fibrillary acidic protein (GFAP) in homogenates of frontal and temporal cortex and elevated phosphofructokinase (PFK) and GFAP in astrocytes from the same brain areas. The data extend previous findings on an increase in brain PFK specific activity in AD and suggest that the increased activity of some glycolytic enzymes may be, at least in part, the result of the reactive astrocytosis developing in the course of AD.

Periodontitis is associated with a loss of colonization by Streptococcus sanguinis

The aim of this study was to estimate differences in the prevalence of oral streptococcal species in the subgingival biofilm of patients with aggressive periodontitis and of healthy controls. Thirty-three patients with clinical and radiological proof of aggressive periodontitis and 20 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. Samples of the subgingival biofilm were taken with paper points from four teeth of each individual. Alpha- and non-haemolytic, small and catalase-negative colonies were biochemically identified using a rapid ID 32 STREP system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A total of 118 strains of oral streptococci (11 species) were identified and Streptococcus sanguinis was found significantly more often in healthy subjects (P=0.001). Conversely, the absence of S. sanguinis was associated with high values of clinical indices (P=0.001-0.002). Aggressive periodontitis seems to be associated with a loss of colonization of S. sanguinis. Whether or not S. sanguinis offers protection against aggressive periodontitis needs to be determined. Otherwise, there were no significant differences in the distribution of oral streptococcal species in patients and healthy subjects.

Rapid identification of oral anaerobic bacteria cultivated from subgingival biofilm by MALDI-TOF-MS

INTRODUCTION To facilitate the identification of anaerobes cultivated from periodontal disease, whole cell bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was evaluated. METHODS A total of 84 strains (nine reference strains and 75 recent clinical isolates from 33 patients with aggressive periodontitis) previously identified with phenotypic methods were used. All the references and 10 clinical isolates belonging to the same species as the reference strains were genotypically identified by sequence analysis of the 16S ribosomal RNA gene. All the strains were then analyzed using MALDI-TOF-MS. RESULTS The reference strains of anaerobic bacteria used showed characteristic MALDI-TOF-MS spectra with peaks between m/z 2000 and up to about m/z 13,000. On visual inspection, the similarity of spectra produced by strains of a single genus could be recognized. Obvious differences between spectra produced by strains of different species were also easily noticed. The reproducibility of the method was proved by the similarity of spectra belonging to the same species. The spectra of the Prevotella intermedia strains identified with MALDI clustered together and clustered separately from the spectra of Prevotella nigrescens, proving that MALDI-TOF-MS is an accurate method that is capable of separating these two species. The quality of clustering was characterized by calculating an inconsistency coefficient (Mathworks:/Matlab Reference Manual v2007a/, Statistical toolbox). CONCLUSION Our results suggest that MALDI-TOF-MS might become a useful method for the identification of anaerobic bacteria, especially for those that cannot be readily identified by biochemical analysis. It may become an attractive system even for the routine identification of clinical isolates.

Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase

By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose 2,6-bisphosphatase was found to be active in cel-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.

Regulation of Human Recombinant P2X3 Receptors by Ecto-Protein Kinase C

The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X3 receptor. When guanosine 5′-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the α,β-methylene ATP (α,β-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X3 receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the α,β-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for α,β-meATP without altering the EC50 values. Molecular simulation of the P2X3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. α,β-meATP-induced currents through native homomeric P2X3 receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X3 receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X3 receptors.

Splice isoforms of ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in human brain

In human brain we were able to demonstrate sequence diversity of the ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). Six different isoforms of PFK-2/FBPase-2, two of which are identical with the ubiquitous PFK-2/FBPase-2 and the inducible PFK-2, respectively, could be identified. The heterogeneity of human brain PFK-2/FBPase-2 isoforms is generated by alternative splicing. Three hitherto unrecognized exons were detected. The multiple PFK-2/FBPase-2 transcripts encode proteins which differ with respect to their length and to the amino acid composition of the carboxyl-termini. The isoform pattern of ubiquitous PFK-2/FBPase-2 is more complex in human brain than in skeletal muscle and liver.

Fructose-1,6-bisphosphatase genes in animals

A comparison of the amino acid sequences of the liver and muscle fructose-1,6-bisphosphatase (FbPase) isoforms in primates and rodents suggested an ancient duplication event leading to the corresponding genes. We investigated the presence of both genes in the rabbit (order lagomorphs) and in species belonging to further distantly related metazoan taxa. By an analysis of the available complete genomes and proteomes of the nematode Caenorhabditis elegans and of Drosophila melanogaster only one sequence homologous to known FbPases was found in each species. The corresponding mRNAs were characterized by cDNA sequencing. We then carried out reverse transcription-polymerase chain reactions to amplify central fragments of the FbPase cDNAs from liver and muscle of Gallus gallus, Xenopus laevis, and Esox lucius, respectively. Their sequencing revealed that (i) the livers of chicken, frog, and fish contain mRNAs which are closely related to mammalian liver FbPase mRNAs, (ii) chicken muscle contains an mRNA which is most homologous to mammalian muscle FbPase mRNAs, (iii) frog muscle contains both a liver-type and a muscle-type FbPase mRNA, while (iv) in fish muscle no FbPase mRNA could be detected by our approach despite the doubtless presence of the enzyme in this organ. An alignment of the partial amino acid sequences of the different FbPases showed that the residues that are thought to be in contact with the substrate, fructose-2,6-bisphosphate, and Mg(2+) are totally conserved, while some amino acids having contact with adenosine monophosphate were found to vary among several species. The question of what might be the advantage of having more than one gene coding for FbPase per haploid genome is discussed.

Cortical glucose metabolism is altered in aged transgenic Tg2576 mice that demonstrate Alzheimer plaque pathology

Summary. Alzheimers disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of β-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of β-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in β-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding β-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparisonto non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high β-amyloid burden. This results from a reduction in glycolytic activity in β-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.