Age-related accumulation of congophilic fibrillar inclusions in endocrine cells

Summary Intracellular fibrillar congophilic inclusions are well known as neurofibrillary tangles in neurons and as Biondi bodies in choroid plexus epithelial cells. Recently similar amyloid-like inclusions in adrenal cortical cells were described (Eriksson and Westermark 1990). This study on 150 adrenal glands confirms these observations. In our material the age-related accumulation of congophilic inclusions starts earlier (in the sixth decade) and reaches a higher incidence (42.7%). We found similar intracellular inclusions in other endocrine organs, for example in the anterior lobe of the pituitary, in the cells of parathyroid glands and in Sertoli cells. The age-related incidence of these fibrillar inclusions in the pituitary was 68%; the co-incidence with interstitial amyloid deposits was 49.5%. Thus the intracellular accumulation of congophilic fibrils in old age is a widespread phenomenon and occurs not only in neurons but also in endocrine cells (adrenal, pituitary and parathyroid glands) and in active secretory cells (choroid plexus and Sertoli cells).     

Cytogenetic analysis of the Asian Plethodontid salamander, Karsenia koreana: Evidence for karyotypic conservation, chromosome repatterning, and genome size evolution

A cytogenetic analysis, including the karyotype, C-bands, silver-stained nucleolus organizer regions and genome size, was performed on the recently discovered species, Karsenia koreana, the first plethodontid salamander from Asia. The karyotype consists of 14 pairs of bi-armed chromosomes, with no evidence of heteromorphic sex chromosomes. C-banding reveals a concentration of heterochromatin at the centromeres as well as at interstitial locations. The smallest chromosome (pair number 14) has symmetrical interstitial C-bands in each arm, resembling chromosome no. 14 of North American species of its sister group taxon, supergenus Hydromantes. Acomparative analysis of C-band heterochromatin and silver-stained nucleolus organizer regions of Karsenia and other plethodontid genera reveals that chromosomal evolution may have featured chromosome 'repatterning' within the context of conserved chromosome number and shape in this clade. Genome size is correlated with geographic distribution in plethodontids and appears to have important phenotypic correlates as well. The genome size of Karsenia is relatively large, and resembles that of the geographically closest plethodontids from western North America, especially species of the genus Hydromantes. The biological significance of these cytogenetic characteristics of plethodontid salamanders is discussed within an evolutionary context.     

Hyperpolarizing current of the Na/K ATPase contributes to the membrane polarization of the Purkinje cell in rat cerebellum

 The contribution of the Na/K ATPase (pump) current to the polarization of the Purkinje cell has been studied using slices of the rat cerebellum by blocking the pump with dihydro-ouabain (DHO) while recording the membrane potential with microelectrodes in the somata. From our recordings, it appeared that blocking the pump depolarized the Purkinje cells more rapidly than might be expected from shifts in Na⁺ and K⁺ concentrations, suggesting the removal of a hyperpolarizing current. Application of DHO, in the presence of tetrodotoxin (TTX), led to calcium spike firing and plateau-like discharges suggesting activation of voltage-dependent calcium channels in the dendrites. Adding 2 mM Co²⁺ to the medium did not prevent the depolarizations. Removing calcium from the bathing medium containing 2 mM Co²⁺ blocked the spiking activity but DHO application still produced a depolarization. Experiments to measure the current inhibited by DHO indicated that the Na/K pump supplies a constant current of 240 pA. Substitution of the sodium with choline produced a hyperpolarization, during which DHO had no effect on the membrane potential. Substitution of the sodium with lithium produced only a slowly developing depolarization. It is concluded that in the cerebellar Purkinje cell, a continuous sodium ion influx activates the pumps which produce a current that directly contributes to the membrane polarization. Possible pathways for this sodium influx are discussed. Received: 3 April 1997 / Received after revision and accepted: 12 May 1997     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Compilation of published comparative genomic hybridization studies

The power of comparative genomic hybridization (CGH) has been clearly proven since the first paper appeared in 1992 as a tool to characterize chromosomal imbalances in neoplasias. This review summarizes the chromosomal imbalances detected by CGH in solid tumors and in hemopathies. In May of 2001, we took a census of 430 articles providing information on 11,984 cases of human solid tumors or hematologic malignancies. Comparative generic hybridization has detected a number of recurrent regions of amplification or deletion that allows for identification of new chromosomal loci (oncogenes, tumor suppressor genes, or other genes) involved in the development, progression, and clonal evolution of tumors. When CGH data from different studies are combined, a pattern of nonrandom genetic aberrations appears. As expected, some of these gains and losses are common to different types of pathologies, while others are more tumor-specific.     

Automated cytofluorometry of solid malignant tumors after cell dispersal by means of intraperitoneal cultivation of cell complexes in young rats (author's transl)]

The use of isolated cells is a basic requirement besides data processing techniques to obtain optimum results with the automated flow cytofluorometry. Several techniques have been employed in order to get cell dispersal. But the claim for reproducible and representative cell samples cannot exactly be fulfilled: gross parts of cells are damaged by any dispersal method applied on solid tissues. Therefore we want to introduce a special method for cell isolation, which avoids mechanical and chemical damage of the treated cells. For this the intraperitoneal tolerance of growing tumor cells in young animals was used, in the species of which solid growth of such tumor cells is established. The investigations reported here were carried out with the Walker-tumor 256 on the rat as a model for solid tumor growth. Our interest was focused on the determination of cellular CNA distribution in tumor cells and contaminating somatic cells grown in the ascitic fluids. Material and Methods: Walker-tumors are permanently cultivated on Sprague-Dawley rats. The solid tumors (1 cm diameter) were removed under sterile conditions, cut with scissors, and resuspended in 0.9% saline. Ten rats (37 days old, body weight 90-120 g) had received an intraperitoneal injection of 1 ml Liquemin (5000 Roche). One million tumor cells could be obtained by such a procedure. After spontaneous sedimentation of cells in order to reduce the number of erythrocytes, the cells were processed and fixed according to Trujillo and Van Dilla (1972), digested with heated RNase (1 mg/ml tris-buffer; 0.18 M and pH 7.5), pepsin treated, and then stained with ethidium bromide (40 mug/ml tris-buffer) for 30 min in the cold. After rinsing with distilled water several times the cells were resuspended in buffer and measured within an hour immediately after passing a 10 mum filter to avoid cell aggregates...     

Absence of fibula and ulna with oligodactyly, contractures, right-angle bowing of femora, abnormal facial morphology, cleft lip/palate and brain malformation in two sibs: a possibly new lethal syndrome

Absence of fibulae, and unilateral absence of ulna, associated with lateral oligodactyly, and rectangular bowing of the femora are the prominent features of a lethal syndrome studied in two children of related parents. Other malformations are cleft lip and palate and posterior midline abnormalities of the brain. One similar though not identical MCA-syndrome was reported. The literature is reviewed with respect to ulno-fibular malformations.     

Abeta-degrading endopeptidase,neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.