Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.     

Interleukin-6 administration has no acute hemodynamic or hematologic effect in the dog

To investigate the possible hemodynamic effects of interleukin-6 (IL-6), a single dose of 15 mcg/kg of recombinant IL-6 isolated from Escherichia coli was injected intravenously in six pentobarbital-anesthetized dogs. After 30 min, saline infusion was performed to maintain the pulmonary artery balloon-occluded pressure at baseline level. The animals were observed for up to 5 hours. No other hemodynamic alteration was observed than a gradual decline in cardiac output attributed to anesthesia. Hematologic variables, blood glucose, and total serum proteins were also constant. IL-6 levels were markedly elevated in the blood, but no tumor necrosis factor activity was detected. Thus a primary role for IL-6 in the early cardiovascular alterations associated with septic shock seems unlikely.     

Enhancement of Endogenous Release of Glutamate and γ-Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long-Term Potentiation

The effect of long-term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and gamma-aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K(+)-induced Ca(2+)-independent release components of these amino acids. In contrast, K(+)-induced Ca(2+)-dependent release of both glutamate and GABA increased approximately 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.     

Effect of Quinine on Autoreceptor-Regulated Dopamine Release in the Rat Striatum

In vivo brain microdialysis was used to examine the role of potassium channel activation in dopamine (DA) autoreceptor function in the striatum of freely moving rats. Local application of the D2 receptor agonists quinpirole or N-0437 through the dialysis probe significantly reduced extracellular concentrations of DA. Local application of the D2 antagonist (-)-sulpiride produced significant increases in DA. Local perfusion with quinine, a K+ channel blocker, completely blocked the (-)-sulpiride-induced increases in DA but did not affect the DA agonist-induced decreases. (-)-Sulpiride completely blocked the effect of quinpirole on DA both in control and in quinine-treated animals. At the highest dose used, quinine caused a large transient increase in extracellular DA. Local application of tetrodotoxin or infusion of Mg2+ in the absence of Ca2+ did not prevent this quinine-induced transient increase in extracellular DA. These results demonstrate that DA autoreceptors in the striatum regulate DA release in awake, behaving animals. Local application of (-)-sulpiride increases DA levels by blocking the tonic activation of autoreceptors by endogenous DA. Quinine blocks the neuroleptic-induced increase in DA, perhaps by preventing the K+ channel opening that would normally accompany endogenous autoreceptor activation. The fact that exogenously applied DA receptor agonists can decrease extracellular DA levels in the presence of quinine suggests that they may be acting at extrasynaptic autoreceptors that are not tonically active in vivo. The effect of DA agonists on this site is via a DA receptor because it is blocked by (-)-sulpiride. However, this receptor does not appear to be coupled to a quinine-sensitive potassium channel.     

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

In vitro propagation and cytology of wild yams,Dioscorea abyssinica Hoch. and D. mangenotiana Miège

Two wild yams of West Africa, Dioscorea abyssinica Hoch, and D. mangenotiana Miège were micropropagated from nodal cultures. Both species produced 4–5 nodes per each node cultured. The size of nodal cuttings was critical, segments shorter than 0.5 cm being less suitable for micropropagation. The number of nodes produced was constant even after 5 cycles of subculture; however, D. abyssinica continuous subculture decreased propagation efficiency, resulting in a reduced number of reculturable nodes at each cycle. In D. mangenotiana, the decrease in multiplication efficiency affected both the number of total and reculturable nodes. Large-sized microtubers were induced on nodal segments maintained under 8-h daylength in both species. In D. abyssinica, however, microtubers were induced on media containing 20, 40, 60 and 80 g l^-1 sucrose, whereas in D. mangenotiana only 40 and 60 g l^-1 sucrose favoured tuberization. Cytological studies confirmed that the chromosome number of D. abyssinica was 2n=40, although a high incidence of cytochimerism and cells with 2n=38 were observed in root meristems. In D. mangenotiana clones, the chromosome number was 2n=40, as against 2n=72 and 2n=80 reported in literature. This species also displayed karyological stability.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - PPF photosynthetic photon flux