Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.     

Effect of amiloride on electrolyte concentrations and rubidium uptake in principal and mitochondria-rich cells of frog skin

The role of mitochondria-rich cells (MR cells) in transepithelial Na transport was investigated by determining electrolyte concentrations and Rb uptake in individual cells of frog skin epithelium using electron microprobe analysis. Measurements were performed under control conditions and after blocking the transepithelial Na transport with amiloride. Under control conditions, Na and Cl concentrations of MR cells scattered much more than those of principal cells and ranged from a few up to more than 30 mmol/kg wet weight. Rb uptake from the basal side into individual MR cells also showed a large variation and was, on the average, much less pronounced than into the principal cells. In principal cells, amiloride reduced the Na concentration and Rb accumulation. In contrast, no effect was observed upon electrolyte concentration and Rb uptake of MR cells. Rb uptake was correlated to the Na concentration of MR cells both under control conditions and after amiloride. It is concluded that, in contrast to the principal cells, MR cells are not involved in amiloride-sensitive transepithelial Na transport and that their Na/K-pump activity is very low.     

Persistent infection of human lymphoid and myeloid cell lines with herpes simplex virus.

Herpes simplex virus (HSV) type 1 replicated and persisted in human T, B, and myeloid cell lines with different patterns of viral replication and various effects on cell growth. T cell line CEM supported the replication of HSV for over 400 days without detectable differences in cell growth as compared with uninfected cells. HSV persisted in B cell line NC37 and myeloid cell line K562 for up to 222 and 374 days, respectively, but led to a significant decrease in the number of viable cells by 7 weeks of infection. The average number of cells producing infectious virus was very low in these cell lines (range, 0.5 to 2.7+) compared with a larger proportion of cells exhibiting HSV antigens by immunofluorescence (range, 24 to 58%). In contrast, null cell line LAZ 221 failed to replicate HSV even though the viral infection led to a cessation of cell growth.     

Die durch Argas (Persicargas) persicus-Larven bedingte Paralyse der Hühner

Zusammenfassung Bei der durch Argas (Persicargas) persicus-Larven bedingten Zeckenparalyse der Hühner handelt es sich um eine generalisierte Affektion des peripheren Nervensystems, wobei es zu einer funktionellen Schädigung der schnell leitenden Nervenfasern kommt.Bei 12 Tieren mit schwersten Paralysen waren die maximalen motorischen Nervenleitungsgeschwindigkeiten, bei gleichzeitig erforderlicher Erhöhung der motorischen Schwellen- bzw. Supramaximalreize, auf 55% herabgesetzt sowie die Reizantwortpotentiale aus der distalen Muskulatur auf 15–30% abgefallen. Die distalen Überleitungszeiten verlängerten sich dabei nur geringfügig. Bei repetitiver distaler Nervenreizung kam es zu einem deutlichen Amplitudenabfall. 3 Tage nach klinischer Restitution hatten sich die neurophysiologischen Veränderungen noch nicht zurückgebildet.Bei 8 Tieren mit mittelgradigen Paresen war die Schädigung des peripheren Nervensystems nicht so stark ausgeprägt. Die maximalen motorischen Nervenleitungsgeschwindigkeiten waren nur auf 70% vermindert, während die motorischen Schwellen- und Supramaximalreize leicht erhöht waren. Die Werte für die Amplituden der Summenpotentiale waren im Vergleich mit den schwerst gelähmten Tieren bedeutend größer, die distalen Latenzzeiten blieben dagegen unverändert. Bei repetitiver Nervenreizung verminderten sich die Amplituden der Antwortpotentiale nur geringfügig. 3 Tage nach der klinischen Rekonvaleszenz waren bei allen Tieren die neurophysiologischen Veränderungen, bis auf eine noch bestehende Verminderung der Summenpotentiale, wieder völlig zurückgebildet.

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Tick paralysis in chickens caused by Argas (Persicargas) persicus-Larvae

Summary In tick paralysis caused by Argas (Persicargas) persicus-larvae a generalized affection of peripheral nervous system especially of fast conducting nerve fibres was found.In 12 animals with severe paralysis maximal motor conduction velocity slowed down to 55% of the initial value and the amplitudes of evoked compound muscle action potentials decreased to 15–30% of the initial value. Simultaneously motor threshold resp. supramaximal stimuli to the nerves were increased. Distal latencies changed only a little. By repetitive distal nerve stimulation muscle action potential amplitudes showed a decrement. Three days after clinical restitution neurophysiologically an affection of peripheral nerves to a small extent could be shown. The affection of peripheral nerves in 8 animals with moderate pareses was less pronounced. The reduction of motor conduction velocity went down only to 70% of the initial value whereas threshold and supramaximal stimuli were only slightly increased. There were in comparison with the severe paralyzed animals higher amplitudes of evoked compound muscle action potentials. Distal latencies remained unchanged. By repetitive nerve stimulation only a small decrement of muscle action potential amplitudes could be stated. Three days after clinical restitution neurophysiologically only a slight reduction of the amplitudes of the evoked muscle action potentials could be measured.

     

Serum-induced chronic pancreatitis

Summary Clinical research into patients with idiopathic chronic pancreatitis points to a possible immunopathogenetic process in a number of cases.In order to examine the behaviour between the exocrine pancreas under the influence of anti-rat-pancreas immune serum produced in rabbits, a 1.00 ml immune serum is administered once a week over a maximum 26 week period into Wistar-rats by intraperitoneal injection. By electrone-microscopy a much reduced production of enzymes apparently takes place, though to differing extent. There is also destruction of the basal membrane of acinocytes; the production of interstitial oedema, the new formation of collagen fibres and the proliferation of connective tissue cells. Under a conventional light microscope the first changes become noticeable after 8-12 weeks of study. These take the form of localised cell decay, deterioration and lysis of acinocytes; and an increasing non-specific inflammation. There is also the new formation of connective tissue. After 20–26 weeks the exocrine pancreas is characterised by reduction of parenchyma, acino-ductal metaplasia, chronic inflammatory infiltrates of differing density, fibrous and irregular calibres of the smaller and larger ducts.The findings are almost identical to the structural changes of chronic idiopathic pancreatitis in human beings. The results support the view of an immuno-pathologic aetiology for human chronic idiopathic pancreatitis.Supported by Deutsche Forschungsgemeinschaft     

Pattern and persistence of the epitope-specific IgM response against human cytomegalovirus in renal transplant patients.

The humoral immune response against human cytomegalovirus (HCMV) was evaluated in immunocompromised patients by Western blotting (WB) based on recombinant viral envelope (gB and gH) and tegument (pp150 and pp65) proteins. Three groups of patients were investigated: (a) 74 renal transplant recipients; (b) 24 hemodialysis patients, both groups without clinical evidence of viral infections; and (c) 19 renal transplant patients with manifest HCMV infections. The results obtained suggest that (i) the WB is considerably more sensitive, recognizing the HCMV-specific IgM response rather than the enzyme-linked immunosorbent assays. An IgM response was detected in one-third of all clinically asymptomatic renal patients. (ii) The virus-specific IgM response is primarily directed against the pp150 epitope. (iii) In patients with clinically manifest HCMV disease, additional IgM reactivities are most frequently directed against the glycoprotein B epitope. (iv) The severity of HCMV infections correlates with the extent of the IgM antibody response, i.e. with the number of specific epitopes involved. (v) After transplantation, IgM reactivity and its epitope-specific pattern persist for years.