Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.     

A synthetic strand of cardiac muscle: its passive electrical properties

The passive electrical properties of synthetic strands of cardiac muscle, grown in tissue culture, were studied using two intracellular microelectrodes: one to inject a rectangular pulse of current and the other to record the resultant displacement of membrane potential at various distances from the current source. In all preparations, the potential displacement, instead of approaching a steady value as would be expected for a cell with constant electrical properties, increased slowly with time throughout the current step. In such circumstances, the specific electrical constants for the membrane and cytoplasm must not be obtained by applying the usual methods, which are based on the analytical solution of the partial differential equation describing a one-dimensional cell with constant electrical properties. A satisfactory fit of the potential waveforms was, however, obtained with numerical solutions of a modified form of this equation in which the membrane resistance increased linearly with time. Best fits of the waveforms from 12 preparations gave the following values for the membrane resistance times unit length, membrane capacitance per unit length, and for the myoplasmic resistance: 1.22 plus or minus 0.13 x 10-5 omegacm, 0.224 plus or minus 0.023 uF with cm-minus 1, and 1.37 plus or minus 0.13 x 10-7 omegacm-minus 1, respectively. The value of membrane capacitance per unit length was close to that obtained from the time constant of the foot of the action potential and was in keeping with the generally satisfactory fit of the recorded waveforms with solutions of the cable equation in which the membrane impedance is that of a single capacitor and resistor in parallel. The area of membrane per unit length and the cross-sectional area of myoplasm at any given length of the preparation were determined from light and composite electron micrographs, and these were used to calculate the following values for the specific electrical membrane resistance, membrane capacitance, and the resistivity of the cytoplasm: 20.5 plus or minus 3.0 x 10-3 omegacm-2, l.54 plus or minus 0.24 uFWITHcm-minus 2, and 180 plus or minus 34 omegacm, respectively.     

Alterations in inositol phosphate production during oxidative stress in isolated hepatocytes

The level of inositol phosphates was measured in rat hepatocytes treated with 2-methyl-1,4-naphthoquinone (menadione) or tert-butyl hydroperoxide, which cause Ca2+ mobilization from intracellular stores and an increase in cytosolic free Ca2+ concentration. Although neither agent produced any apparent changes in the resting level of inositol phosphates, pretreatment of hepatocytes with either menadione or tert-butyl hydroperoxide, as well as with several sulfhydryl reagents, markedly inhibited the increase in inositol phosphates induced by both hormonal and nonhormonal stimuli. Addition of dithiothreitol to menadione- or tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness to extracellular stimuli. Our findings suggest that the inhibition of the inositol phosphate response by menadione and tert-butyl hydroperoxide occurs through the modification of critical sulfhydryl group(s) and that the alterations in intracellular Ca2+ homeostasis occurring during the metabolism of menadione and tert-butyl hydroperoxide in hepatocytes are not mediated by inositol phosphates.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Improvement of artificial endocrine pancreas (Biostator; GCIIS) performance combining feedback controlled insulin administration with a pre-programmed insulin infusion

It is well known that the glycemic control accomplished by the artificial endocrine pancreas (Biostator; GCIIS), requires an excessive insulin consumption. In order to improve the Biostator performance, we have studied 5 insulin dependent diabetic patients on anticipated automatic pre-programmed insulin infusion starting at the beginning of breakfast. Such an operating method allowed us to obtain a significant reduction in insulin requirements (from 17,3 +/- 2,2 U to 13,3 +/- 2,2 U), and a better glycemic control (MBG 98,7 +/- 5,4 vs 111,1 +/- 5,6; M 2,34 +/- 0,9 vs 6,96 +/- 1,7; delta G 43,8 +/- 3 vs 73,4 +/- 9,7), as compared to those observed under conventional feed-back control in relation to breakfast.     

Critical role of sulfhydryl group(s) in ATP-dependent Ca2+ sequestration by the plasma membrane fraction from rat liver

The ATP-dependent sequestration of Ca²⁺ by the plasma membrane fraction from rat liver is stimulated by reduced glutathione and dithiothreitol and inhibited by diamide and t-butyl hydroperoxide. The inhibitory effect on Ca²⁺ sequestration by the oxidizing agents is prevented in the presence of the thiols. Our results therefore suggest that free sulfhydryl group(s) may be critical for the activity of hepatic plasma membrane Ca²⁺ translocase, and that inhibition of this activity by the oxidation of such group(s) may contribute to the perturbation of Ca²⁺ homeostasis during oxidative stress.     

Long-term effects of nicotine on the forced swimming test in mice: an experimental model for the study of depression caused by smoke

Large evidence showing an association between depression and tobacco smoking is known. Nicotine is the active chemical responsible for smoking addiction, and its withdrawal may induce in smokers greater sensitivity to stress. Our aim has been to investigate the links between tobacco addiction and depression by studying the long-term effects of repeated administration of nicotine followed by dependence, to forced swimming test, serotonin content and 5-HT(1A) expression in diencephalon. Dependence has been induced by daily subcutaneous injection in mice of nicotine (2mg/kg four injections daily) for 15 days and assessed after nicotine withdrawal with an abstinence scale; control animals received daily subcutaneous injection of saline for the same period. Experiments on forced swimming test have been carried out at t=0 (last day of nicotine or saline treatment), and 15, 30, 45 and 60 days after saline or nicotine withdrawal. Both control mice and nicotine mice have been pre-treated with oral 5-hydroxy-tryptophan (12.5-50mg/kg), precursor of serotonin, before forced swimming test. Nicotine mice have shown on forced swimming test a significant increase of immobility time compared to control mice. This increase was not evident in nicotine mice treated with 5-hydroxy-tryptophan and treatment with the selective serotonin receptorial antagonist WAY 100635 (WAY) abolished 5-hydroxy-tryptophan effects. Evaluation of diencephalic serotonin, performed at t=0 showed an increase of diencephalic serotonin content, while serotonin measured 15, 30, 45 and 60 days after nicotine withdrawal, was significantly reduced in nicotine mice compared to control mice. Western blot analysis showed a great reduction of 5-HT(1A) receptor expression in nicotine mice measured at t=0 (last day of treatment) and at 15 and 30 days after nicotine withdrawal compared to control mice. Our results show that (i) behavioural alterations estimated with forced swimming test and (ii) changes in diencephalic serotonin content and 5-HT(1A) receptor expression, are present since nicotine is withdrawn even after a long time, suggesting a role of serotonin in mood disorders eventually occurring following smoking cessation.     

Differential levels of soluble endoglin (CD105) in myeloid malignancies

Angiogenesis contributes to disease progression in solid and hematopoietic malignancies, and endoglin (CD105), a component of the transforming growth factor (TGF)-beta receptor complex, is a powerful marker of neovascularization. Elevated amounts of soluble CD105 (sCD105) have been recently identified in selected solid tumors but no data are available on sCD105 in hematopoietic malignancies. Therefore, levels of sCD105 were investigated in sera of patients with acute myeloid leukemia (AML) (n = 10) or chronic myeloproliferative disorders (CMD) (n = 28), and correlated with those of soluble TGF-beta(1) (sTGF-beta(1)). Dot blot assay detected higher amounts of sCD105 (P < 0.05) both in AML (4.34 +/- 2.62 OD/mm(2)) and in CMD (3.71 +/- 2.09 OD/mm(2)) patients than in healthy subjects (n = 14, 2.38 +/- 1.18 OD/mm(2)). Instead, enzyme-linked immunosorbent assay (ELISA) identified (P < 0.05) lower and higher levels of sTGF-beta(1) in AML (32,017 +/- 1,900 pg/ml) and CMD (60,700 +/- 19,200 pg/ml) patients, respectively, compared to healthy individuals (n = 11, 47,173 +/- 5,443 pg/ml). In essential thrombocythemia (ET) patients with thrombotic episodes, levels of sCD105 were lower (P < 0.05) compared to patients without thrombotic complications, and inversely correlated with those of sTGF-beta(1) (r = 0.94). Conversely, amounts of sCD105 directly correlated with levels of sTGF-beta(1) (r = 0.74) in ET patients without thrombotic events. Our results show that high levels of sCD105 are present in myeloid malignancies that are characterized by a high cellular proliferation rate, and suggest that an altered balance between sCD105 and sTGF-beta(1) might favor disease progression and clinical complications.     

Mitochondrial Respiratory Dysfunction in Familiar Parkinsonism Associated with PINK1 Mutation

In the present study mitochondrial respiratory function of fibroblasts from a patient affected by early-onset Parkinsonism carrying the homozygous W437X nonsense mutation in the PINK1 gene has been thoroughly characterized. When compared with normal fibroblasts, the patient’s fibroblast mitochondria exhibited a lower respiratory activity and a decreased respiratory control ratio with cellular ATP supply relying mainly on enhanced glycolytic production. The quantity, specific activity and subunit pattern of the oxidative phosphorylation complexes were normal. However, a significant decrease of the cellular cytochrome c content was observed and this correlated with a reduced cytochrome c oxidase in situ-activity. Measurement of ROS revealed in mitochondria of the patient’s fibroblasts enhanced O₂^•− and H₂O₂ production abrogated by inhibition of complex I. No change in the glutathione-based redox buffering was, however, observed. Special issue article in honor of Anna Maria Giuffrida-Stella.