Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility.

Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2, and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis.

Three catalytic sites in mitochondrial ATPase.

Kinetic data obtained after determining the hydrolytic activity of ATPase from rat liver in preparations where the enzyme had been purified, or in mitochondria, strongly suggest the existence of three different catalytic sites with different affinity for the substrate. The results obtained when measuring the ATPase activity at different substrate concentrations, and in the presence of the inhibitors KOCN or KSCN, or of the activators dinitrophenol and bicarbonate, show that the binding of these compounds to a regulatory site or sites affects in a different degree the hydrolytic activity of each catalytic site.

Determination of arginase activity in macrophages: a micromethod.

We propose a modification of Schimke's method for urea determination as a valuable micromethod for measuring arginase in activated macrophages. The method exhibits the following advantages: (a) it uses small amounts of samples (approximately 25,000 macrophages per assay); (b) it does not interfere with other related metabolites that are also present in the activated macrophage such as citrulline or arginine; (c) saturating concentrations of the substrate arginine can be used; and (d) it is much more sensitive than Schimke's method and can detect small amounts of urea, in the order of 0.02 mumol.

Inhibition of phosphatidylcholine synthesis precedes apoptosis induced by C2-ceramide: protection by exogenous phosphatidylcholine.

Cerebellar granule neurons in primary culture underwent apoptosis when exposed to C2-ceramide. Addition of exogenous phosphatidylcholine (PtdCho) resulted in a dose-dependent full prevention of neuronal death. Exogenous PtdCho also prevented apoptosis induced by farnesol, N-oleoylethanolamine, and sphingomyelinase, but did not prevent apoptosis induced after lowering the potassium concentration in the medium to non-depolarizing levels. Moreover, C2-ceramide inhibited labeling of [32P]PtdCho in cells incubated with [32P]orthophosphate, with the same potency to that causing apoptosis. Although cell viability did not decrease during the first few hours, inhibition of PtdCho synthesis was already patent after a 1 h exposure to C2-ceramide. Taken together, these results strongly suggest that inhibition of PtdCho synthesis constitutes one of the primary events by which C2-ceramide triggers apoptosis in cerebellar granule neurons.

[First evaluation of three years of assessment of medical activity within the framework of PMSI in cancer prevention centers]. / Premier bilan de trois années de recueil de résumés standardisés de sortie dans le cadre du PMSI dans un centre de lutte contre le cancer.

In June 1982, the Direction of the French Hospitals introduced the project for the medicalisation of the system of information (PMSI) derived from the American Fetter system. The aim of this project was the collection of basic data necessary to the establishment of a national minimum data set base (MDS). Since 1985, a french regional cancer center, the Foundation Bergonié, has collected MDS by computer. Now, we give the results of the analysis concerning 28,379 MDS collected from 1986 to 1988. However, 11,127 (39%) MDS have not yet been analyzed by PMSI as they are connected with external activity or ambulatory care. It is possible to form a diagnosis related group (DRG) from the MDS to analyze the management of hospitals and utilization of means. 279 DRG were formed from 12,252 MDS, in our Institute. In fact, only 30 DRG with a sufficient number of MDS (over 100) were studied. This descriptive study shows that a few DRG maintain homogeneity in terms of length of hospitalisation especially in the surgical sector. It seems necessary to incorporate new criteria for MDS in order to improve the study of DRG; however groups sufficiently large for statistical analysis must be maintained.

Protonmotive force and photophosphorylation in single swollen thylakoid vesicles.

Swollen vesicles generally 40 micron in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin-luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a delta pH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.

Protonmotive force in giant mitochondria.

The accompanying communication [Eur. J. Biochem. 141 (1984) 1-4] indicates that the microinjection of the pH fluorescent indicator pyranine (8-hydroxy-1,2,6- pyrenesulfonate ) into giant mitochondria or mitoplasts does not affect their ability to carry out oxidative phosphorylation. The dye can therefore be used as a quantitative indicator of internal mitochondrial pH. We found that activation of metabolism in rotenone-inhibited giant mitochondria by the addition of succinate produces an internal pH change corresponding to a pH shift of 0.3 to the alkaline range, approximately the same value found previously for conventional rat liver mitochondria.

Properties of arginase immobilized in a fibrin clot.

Rat liver arginase was covalently trapped in a fibrin clot. Among the physicochemical properties of the enzyme studied were Mn2+ requirement, pH behavior, temperature and time stability, effect of denaturing agents, and kinetic properties. The immobilized arginase showed the same substrate affinity as soluble arginase, but had higher stability at room temperature, was more resistant to denaturation, and had a higher catalytic activity at physiological pH. The properties so far examined may enhance the use of immobilized arginase in cancer therapy.

Kinetics of manganese reconstitution and thiol group exposition in dialyzed rat mammary gland arginase.

1. Rat mammary gland arginase is a metallo-enzyme dependent on Mn2+, which can be only partially substituted by Cd2+. 2. Reconstitution of the activity of dialyzed arginase by manganese is a two-phase process; the second phase is independent of the cation concentration, with a half-time recovery (t1/2) of 10.77 min. 3. The apparent Km for Mn2+ is 280 microM and 10.5 microM for enzyme dialyzed for 24 and 72 hr, respectively. 4. Treatment with 5 mM EDTA at pH 6 totally inhibits enzyme activity, which is reconstituted by Mn2+. 5. Results obtained with iodoacetamide treatment suggest the existence of sulphydryl groups accessible only when the enzyme is dialyzed.

Kinetics and inhibition by some aminoacids of lactating rat mammary gland arginase.

Some kinetic and regulatory properties of lactating rat mammary gland arginase were studied. At pH 7.4, i.e. at near-physiological conditions, there was evidence of inhibition by excess of substrate, with a Km value of 9.5 mM, slightly lower than the value of 18 mM observed at pH 9.8 (maximum enzyme activity). A study was also made of the effects of proline, ornithine, lysine and certain branched-chain aminoacids on enzyme activity: lactating rat mammary gland arginase was strongly and competitively inhibited by lysine, ornithine and valine, with Ki values of 1.2 mM, 1.1 mM and 3.6 mM, respectively. Other aminoacids (proline, isoleucine and leucine) also inhibited lactating rat mammary gland arginase, although to a lesser extent.

Trypsin digestion of arginase: evidence for a stable conformation manganese directed.

1. Controlled tryptic digestion of native arginase from rat liver suggests that Mn2+ promotes a stable conformation as shown by the following features. 2. An 18-fold increase in the half-life of arginase activity in the presence of Mn2+ is produced. 3. The stability of subunit B of arginase is increased in the presence of Mn2+ as revealed by SDS-PAGE during the time-course of trypsin cleavage. 4. The different digestion products of arginase with and without Mn2+ appearing during the time-course of tryptic treatment. 5. Different activity/bands protein ratio at any time of the tryptic digestion in the incubation mixtures, with and without Mn2+, are apparent.

The effect of antimycin A on mouse liver inner mitochondrial membrane channel activity.

In a patch-clamp study, we found antimycin A in low (1-2) microM concentrations decreased the open probability of the multiple conductance channel activity and the approximately 110 picosiemens channel of the inner mitochondrial membrane (for a review of mitochondrial channels see Kinnally, K. W., Antonenko, Yu. N., and Zorov, D. B. (1992) J. Bioenerg. Biomembr. 24, 99-110). Higher antimycin A concentrations (e.g. 10 microM) facilitated multiple conductance channel opening. These effects were reversible, and the binding site(s) are probably distinct from those responsible for the inhibition of the electron transport chain, since the latter are virtually irreversible. A model with two closed and two open states is presented for the approximately 110-picosiemens activity.

MCC and PSC, the putative protein import channels of mitochondria.

All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in the cytoplasm and imported into the organelle. Water-filled channels are integral to the process of translocating proteins since channels can provide an aqueous pathway through the hydrophobic environment of the membrane. The MCC (multiple conductance channel) and PSC (peptide-sensitive channel) are two high-conductance channels previously identified in electrophysiological studies of mitochondrial membranes. MCC and PSC are the putative pores of the import complexes of the inner and outer membranes, respectively. The genetic, biochemical, and biophysical evidence regarding these assignments are summarized herein. These findings support the identification of MCC and PSC as the protein import channels of mitochondria.

Mitochondrial channel activity studied by patch-clamping mitoplasts.

Patch-clamping mitoplasts, we have observed a complex pattern of conductance transitions. This report discusses primarily the 45, 120-150, 350, and 1,000 pS transitions.

Physico-chemical properties of hepatocyte plasma-membrane-bound arginase.

Rat liver plasma-membrane-bound arginase was investigated in order to obtain data regarding its physico-chemical properties. Arginase bound to plasma membrane presented a specific activity of 0.74 +/- 0.09 IU/mg for the fully-activated enzyme, the pH of maximum activity being 9.8. Maximum stability was recorded at two pH values, 7 and 10.5 respectively. Mn2+ activated the enzyme, while Cu2+ and Zn2+, and to a lesser extend Co2+, showed a strong inhibitory effect. Ca2+ and Mg2+ had no effect at the concentrations assayed. The influence of temperature was studied in the presence and in the absence of Mn2+. The enzyme was stable up to 65 degrees C in both cases. Membrane- bound arginase showed an activation energy of 11.5 +/- 1.4 Kcal/mol between 20 and 40 degrees C, and 13.3 +/- 2.5 Kcal/mol between 40 and 60 degrees C. The Q10 for the same temperature ranges were 1.78 and 1.9 respectively. The membrane-bound enzyme presented two different Michaelis constants, one with high affinity (2.05 +/- 0.73 mM) and the other with low affinity for arginine (130 +/- 27.2 mM). Solubilized arginase showed very similar values. Among all the structural analogous assayed, only L-canavanine proved to be substrate for arginase, with and L-arginine/L-canavanine hydrolysis ratio of 5.8 +/- 0.28. No reactivity was found between plasma-membrane-bound arginase and anti-rat liver arginase antibodies raised in rabbits.

An arginine regulated gamma-guanidobutyrate ureahydrolase from tench liver (Tinca tinca L.).

A gamma-guanidobutyrate ureahydrolase isolated from tench liver has been characterized. Some of its physicochemical properties like pH effect and thermal stability resemble those of arginases, however it shows some peculiarities that makes it different from arginases and other amidino hydrolases. Thus cation requirement is not as strong as in arginases, and the Km value for gamma-guanido-butyric acid (230 +/- 25 mM) is shifted to a lower value (45 +/- 5 mM) by 5 mM arginine. The possible regulatory role of arginine on gamma-guanidobutyrate ureahydrolase activity is discussed.

Assay of ATP synthesis using single giant mitochondria.

Two assays of the capacity of single giant mitochondria or mitoplasts to phosphorylate ATP from Pi and ADP have been developed. One depends on the placement of a single mitochondrion next to a glycerinated myofibril, by micromanipulation. With the appropriate controls, contraction of the myofibril serves as an indication of ATP synthesis. The other assay similarly requires the isolation of one single mitochondrion but the assay of ATP synthesized uses the luciferin-luciferase reaction with a conventional photometric system. With the latter, we found that either impalement with microelectrodes or electrophoretic microinjection of two dyes, Lucifer Yellow CH or pyranine , into the inner space have no effect on the phosphorylative capacity of mitochondria or mitoplasts. The electric potential across the mitochondrial membrane was monitored during the assay and found to be small and generally positive inside.

Parallel induction of nitric oxide and glucose-6-phosphate dehydrogenase in activated bone marrow derived macrophages.

The production of nitric oxide (NO.) and the induction of glucose-6-phosphate dehydrogenase by lipopolysaccharides (LPS) from different sources was studied in bone marrow derived macrophages (BMM phi). NO. production was found to be linked to the induction of glucose-6-phosphate dehydrogenase, suggesting the possible involvement of this enzyme in the cytotoxic mechanism resulting from the release of NO. by activated macrophages.

Immunological identity of the two different molecular mass constitutive subunits of liver arginase.

A detailed understanding of the regulatory mechanisms of arginase in the cell will depend on the clarification of the origin of the two different molecular mass subunits and on the arrangements of them to constitute the native enzyme. Here, we show the immunological recognition of the 39.5 and 37.0 kDa subunits of arginase by antibodies against both subunits. We also find that the subunit stoichiometry (39.5 kDa: 37.0 kDa) present in purified arginase preparations as well as in fresh isolated microsomes and cytoplasm corresponds to 3:1, indicating high prevalence of a constant arrangement of the constitutive subunits of arginase. These findings represent evidence for a limited posttranscriptional or posttranslational modification of only a fraction of the synthesized arginase in liver.

Effect of nucleotides and inhibitory anions on mitochondrial ATPase. Its pH dependence.

The effect of pH on the sensitivity of F1-ATPase as well as mitochondrial ATPase activity to nucleoside diand triphosphates and to inhibitory anions such as cyanate and thiocyanate, has been studied. The results obtained show that nucleotides could act as activators or inhibitors of the ATPase hydrolytic activity depending on pH, substrate concentration, and binding of the enzyme to the membrane. The effect of those nucleotides which activate the hydrolysis of ATP-Mg2+ was more pronounced beyon the optimum pH corresponding to each of the three catalytic sites of the enzyme, whereas those which are inhibitors had a lower effect above this value. The sensitivity to the inhibitory anions decreased with increasing pH values; the decrease in the inhibitory effect was sharper when approaching the optimum pH value. These data are in agreement with the existence in mitochondrial ATPase of two different regulatory sites, one being specific for binding nucleotides, and another for anions. Both of them showed a different response upon changes of pH.