Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells

This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms. OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay. Nicotine (10⁻⁶ − 10⁻³ M) stimulated significant OLF secretion in rat adrenocortical cells. Acetylcholine (10⁻⁷ − 10⁻⁴ M) and eserine (10⁻⁷ − 10⁻³ M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations. Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion. ACTH (10⁻⁸ M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent. The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion. The α7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion. These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.     

Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.     

Molecular evolution and geographic origins of type 1 human lymphotrophic virus in Colombia detected by RFLP polymorphism

The human T-lymphotropic virus type I (HTLV-I) infection is a public health problem in many endemic areas of Colombia. The subtyping of HTLV-I was based on the analysis of restriction fragment length polymorphisms (RFLP) in 3'LTR proviral DNA. From 31 HTLV-I isolates collected throughout Colombia, a RFLP analysis in a 737 bp 3'LTR fragment was performed. Fifty-eight percent (18/31) were identified as the Cosmopolitan subtype a, 19.4% (6/31) in the West African subtype b, 12.9% (4/31) in the Cosmopolitan subtype b and 9.6% (3/31) in the West African subtype c. The phylogenetic analysis of 3'LTR nucleotide sequences indicated that all the isolates in the current study were in the subgroup B or Japanese, in contrast with the highly divergent isolates from native Amerindians grouped in subgroup a or Transcontinental. The supported hypothesis was that of a post-Columbus introduction of virus represented in the African-American communities of the Colombian South Pacific. Some viral isolates from Colombian native Amerindians exhibited a nucleotide variation compatible with a Paleolithic introduction of the virus. The genetic diversity of HTLV-I in Colombia is complex and probably represents several independent introductions of lymphotropic virus.     

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.     

Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity

The effect of agaric acid as inducer of mitochondrial permeability transition was studied. It was found that: (i) agaric acid (AA) promoted efflux of accumulated Ca2+, collapse of transmembrane potential, and mitochondrial swelling; (ii) these effects depend on membrane fluidity; (iii) ADP inhibited the effect of AA on Ca2+ efflux, and (iv) AA blocked binding of the sulfhydryl reagent, eosin-5-maleimide, to the adenine nucleotide translocase. It is proposed that AA induces pore opening through binding of the citrate moiety to the ADP/ATP carrier; this interaction must be stabilized by insertion of the alkyl chain in the lipid milieu of the membrane.     

Studies on the conformation of protonated DNA

Experiments were made to demonstrate the predominant protonation effects and structural changes of the ordered double helical DNA structure and denatured state of DNA. Spectrophotometric titrations performed at different wavelengths indicate that cytosine can be protonated in the DNA double helical molecule to a high extent without breakdown of the secondary structure. With DNA heat-denatured under severe conditions the protonation of cytosine can be measured at 280, 295, and 300 mμ: the apparent pK value obtained was ～4.6. The protonated double helical conformation of the DNA molecule differs from the unprotonated state, which follows from the decrease of the thermal stability and from changes in the ORD curves. The ORD of a GC-rich DNA indicates a novel Cotton effect with positive rotations at ～260 mμ in 0.02M KCl below pH 4.0 to pH 3.3. The occurrence of the new peak parallels the extent of protonated cytosine measured by the spectrophotometric titrations. It is concluded that the protonated cytosine in the double helical structure is responsible for the difference between the protonated DNA conformation and the native state at neutral pH.     

Altersbedingte Anhäufung von lysosomalen Residualkörpern in neurosekretorischen Zellfortsätzen

Zusammenfassung Im Anschluß an frühere Beobachtungen über die Reaktion von Herringk: orpern auf osmotische Belastung wurden die Veränderungen dieser Strukturen mit zunehmendem Alter studiert. In parallelen färberisch-lichtmikroskopischen und elektronenmikroskopischen Untersuchungen wurden die im Gebiet der Nuclei supraopticus und paraventricularis gelegenen Herringkörper von jungen (3 Monate) und alten (15–20 Monate) Ratten miteinander verglichen. Bei alten Tieren findet man im Lichtmikroskop eine starke Zunahme der durch Neurosekretfärbung darstellbaren Herringkörper und im Elektronenmikroskop eine Füllung dieser Gebilde mit dense bodies und Myelinkörpern. Diese werden als lysosomale Residualkörper gedeutet, die aus den neurosekretorischen Perikarya in die Zellfortsätze abgeschoben werden und sich dort mit zunehmendem Alter anhäufen. Die fraglichen Zellfortsätze werden aufgrund von morphologischen und topographischen Gesichtspunkten als Dendriten angesehen.

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Age-dependent accumulation of lysosomal residues in neurosecretory cell processes

Summary In continuation of previous investigations on the reaction of neurosecretory Herring bodies to osmotic stress, the alterations of these structures with increasing age were studied. The Herring bodies located in the vicinity of the supraoptic and paraventricular nucleus of young (3 months) and aged (15–20 months) rats were compared by means of light and electron microscopy. In old rats, the light microscope reveals a strong increase in the number of Herring bodies as visualized by neurosecretory stain whereas the electron microscope exhibits an accumulation of dense bodies and myelinated bodies inside these structures. These are interpreted as lysosomal residual bodies which are transported from the neurosecretory pericarya into the cell processes, thus accumulating with increasing age. Morphological and topographical considerations lead to the assumption that the storage sites are dilatations of dendrites.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Kernzahl und Zellgröße im Fruchtkörperstiel von Coprinus radiatus (Solt) Fr.

Vor der Streckung der Stielzellen von Coprinus radiatus erhöht sich die Zahl der Kerne durch mehrere synchrone Teilungen.