Guy Graff

Evidence for a clathrin-mediated recycling of albumin in human term placenta.

Abstract During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 μM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-β-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.

Alkaline phosphatase isoenzyme pattern in human amniotic fluid is dependent on the level of total activity. Implications in cystic fibrosis diagnosis.

Alkaline phosphatase activities have been examined in 500 consecutive human amniotic fluids obtained at diagnostic paracentesis between 14 and 38 wk of gestation. They were found to have a non-Gaussian, positively skewed distribution, independent of the protein concentrations. Residual activities after heat treatment or in presence of phenylalanine and levamisole allow evaluation of the placental, hepatic and intestinal isoenzyme components. It is shown that the contribution of the intestinal isoenzyme to the total activity is a linear function of the latter. This fact should be taken into account in the enzymatic diagnosis of cystic fibrosis as it is apparent that the part contributed by the intestinal isoenzyme is predictably smaller when the level of total activity is low.

Systemic effects of N, N-dimethyl-paraphenylenediamine hydrochloride on phosphate metabolism in innervated and denervated, slow and fast muscles of the rat

N, N-dimethyl-paraphenylenediamine (DMPPD) hydrochloride increases the inorganic and organic acid-soluble phosphate (Pi and POAS) uptakes in the innervated gastrocnemius muscle of the rat but not in the innervated soleus. In the denervated gastrocnemius muscle, the effects of DMPPD and denervation are not additive, Pi uptake being even lower than on the controlateral innervated side. It is suggested that DMPPD acts only on the innervated fast fibre (white fibre, type II anaerobic fibre), as far as the permeability to Pi is concerned. Histological evidence of a severe myopathic process affecting slow and fast fibres, irrespective of denervation has been documented. Severe degenerative changes were still manifest after a 20-day DMPPD treatment. There is no obvious relation between the biochemical alterations in phosphate metabolism and the morphological lesions. The increased Pi and POAS uptakes observed in the innervated gastrocnemius and soleus muscles during generalized convulsions induced by DMPPD are independent of a direct drug action on the muscle fibre.

Réparation du nerf périphérique: intérêt des colles biologiques et de la suture épipérineurale dans les interventions tardives: Etude expérimentale chez le rat

In order to approximate as close as possible genuine clinical conditions, the sciatic nerve of the rat was divided and then repaired after a delay of one or seven days either by application of a biological glue or by an epiperineural suture technique. The metabolic activity of the sciatic nerve Schwann cells - whether located in the distal or the proximal ends - and that of the (fast acting) white gastrocnemius and (slow acting) red soleus muscle were assessed using 32P-radiolabeled acid-soluble phosphates. Delayed repair, as judged by our biochemical criteria, was equivalent whatever method, biological glue or suture, was used. The frozen and lyophilized forms of the biological glue provided similar results.

SERUM L‐LEUCYL‐β‐NAPHTHYLAMIDE HYDROLASE ACTIVITY IN NORMAL SINGLE AND TWIN HUMAN PREGNANCIES AND IN PREGNANCIES ASSOCIATED WITH FETAL DYSMATURITY

NORMAL human serum has L-aminoacyl+?naphthylamide hydrolase activity which is carried by a protein with a,-globulin mobility in starch gel electrophoresis (Wintersberger and Tuppy, 1960; Page et a/ . , 1961). During normal pregnancy new serum activities appear which are carried by two proteins with mobilities respectively of fast a2 and slow a,-globulins (Page et al., 1961; Glendening et al., 1965). Kleiner and Schram (1966) confirmed these observations by vertical acrylamide gel electrophoresis. The evolution of the total L-leucyl-pnaphthylamide hydrolase (LNAse) activity and of the isoenzyme activities in the maternal serum was followed during the course of normal human pregnancy by Kleiner and Graff (1967). The placental origin of the new enzymatic activities is supported by clinical, biochemical, and histochemical evidence as well as by observations on placental tissue cultures (Page et al., 1961; Semm and Waidl, 1962; Kleiner Wilkin and Snoeck, 1962 ; Kleiner and Wilkin, 1963 ; Sciarra and Long, 1963; Wachstein et al., 1963; Jonek et al., 1965; James, 1966; Bernhard, 1967). Whether the placenta is the only possible source for the serum pregnancy activities remains, however, debatable (Riad, 1962; Beckman et al., 1966; Tuppy and Wintersberger, 1964; Kleiner and Graff, 1966. L-aminoacyl-/?-naphthylamidase activity determinations in several pathological conditions complicating pregnancy showed lowered values in cases of intrauterine death and in the “placental insufficiency” syndrome (Hashimoto, 1961 ; Kleiner et al., 1963; Kleiner and Van Rymenant, 1964). However, these few early observations did not allow definite conclusions. Several publications have since confirmed the value of LNAse determinations in pathological conditions associated with human pregnancy. Lowered values were observed in chronic fetal distress associated with prolonged pregnancy (Cisternino and Messina, 1965) and in “placental insufficiency” (Panella et al., 1966 ; Josephides and Turkington, 1967 ; Babuna and Yenen, 1966a). Increased values were, on the other hand, reported in multiple pregnancy (Sciarra and Burress, 1960; Hashimoto, 1961; Miller et a/ . , 1964; Babuna and Yenen, 1966b). It was therefore hoped that LNAse serum assays would be useful in assessment of the welfare of the pregnancy, and for detection of abnormal cases. The following data are presented to show the significance of lowered LNAse values in pregnancies associated with fetal dysmaturity. Data pertaining to normal twin pregnancies will also be given.

Increased muscle regeneration after repair of divided motor nerve with neuronotrophic factors containing glue

Neuronotrophic factors (NTFs) directed to spinal cord motor neurons were collected in rats within silicone nerve regeneration chambers according to LONGO et al. (1983b). Unilateral addition of NTFs to the fibrin glue used for the repair of divided sciatic nerves improved locally nerve regeneration without affecting the controlateral side. Nerve regeneration was assessed by weight gain of the reinnervated muscles and by radioactive labelling of the acid-soluble phosphate fractions of both nerve Schwann cells and reinnervated muscle cells. Fast gastrocnemius and slow soleus muscles, the motor nerve of which had been repaired with added NTFs, were significantly heavier (21 and 28%) than their controlateral controls, and the metabolic dedifferentiation attendant on post-division nerve repair was less marked. It is suggested that this experimental nerve regeneration model is suitable to test potential nerve-active agents in vivo, under conditions close to the usual clinical setting, with, as ultimate goal, the improvement of the end-results of microsurgical repair of peripheral nerve in man.

Effect of increasing duration of denervation on the rate of entry of inorganic phosphate into rat gastrocnemius muscle.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle, is shown to have no direct relation with either the loss of muscle mass or with the concentrations of the acid-soluble phosphate fractions. It is shown to increase hyperbolically with the time elapsed since the nerve section. The asymptotic value reached after thirty days suggests the presence of a saturable mechanism.

Alkaline phosphatase activities in normal and denervated, slow and fast rat muscles. Bearing on the possible participation of the enzyme in the transmembrane transport of phosphate ions.

Alkaline phosphatase (AP) activities are present in rat gastrocnemius:48.7, plantaris: 68.9, tibialis anterior: 69.1 and soleus: 96.7 nmol phenol. min-1. 100 mg muscles-1. These concentrations are one and two orders of magnitude lower than those observed in duodenum and placenta, but similar to those observed in liver. Response to activators/inhibitors and electrophoretic behaviour assign the muscle AP activities to the rat liver/placenta isoenzyme group. Motor denervation does not affect significantly the total muscle AP content within the first 30 postoperative days, however the concomitant variations in muscle weight are responsible for wide differences in AP concentrations between innervated, denervated and reinnervated muscles. Parallel determinations of radiophosphate uptake and AP activities failed to document a necessary link between the two variables, i.e. between enzyme concentration and phosphate ion transport.

Phosphate metabolism and foetal growth in the rat. I. Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the normal placenta

Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the rat foetus has been studied after intraperitoneal injection of [32P] ortho-phosphate in primigravid females at the 12th day or later stages of gestation. The concentration per unit weight of foetus of the inorganic phosphate (P1) fraction increases markedly with increasing foetal weight; labelling data [inverse relationship between P1 concentration and specific activity, absence of precursor/product relationship between P1 and acid-soluble organic-bound phosphates (POAS)] show this increase to result in part from the formation of a relatively inert metabolic pool, presumably in mineralized tissue. The foetal concentrations of calcium and inorganic phosphate show a strong positive correlation, both increasing markedly with foetal weight. The progressive accumulation of calcium does not, however, account entirely for the rising concentration of inorganic phosphate. The concentration per unit weight of foetus of the POAS fraction remains stable for foetuses smaller than 2 000 mg. In heavier foetuses (greater than 2 000 mg) the POAS concentrations are, with an abrupt transition, distinctly lower, rising however slightly with increasing foetal weight. The concentration per unit weight of foetus of the acid-insoluble organic-bound phosphate (POAIS) fraction decreases slightly with increasing foetal weight. The label uptake per unit weight of foetus of both POAS and POAIS fractions is negatively correlated with increasing foetal weight. The amount and label uptake per whole foetus of the P1, POAS and POAIS fractions are positively correlated with increasing foetal weight. Phosphate transfer to the foetus increases continuously, being maximal at or near birth.

Systemic effects of colchicine on phosphate metabolism in innervated and denervated, slow and fast muscles of the rat

A single systemic injection of 75 micrograms colchicine/100 g body weight increases the permeability to inorganic phosphate of both fast gastrocnemius and extensor digitorum longus muscles and of the slow soleus muscle. In the two fast muscles, there is a significant interaction between colchicine treatment and 5-d- or 30-d- surgical denervation. In the slow soleus muscle there is no interaction between 5-d-colchicine treatment and the initial decrease in phosphate flow due to 5-d-surgical denervation but a significant interaction between colchicine treatment and the secondary increase in phosphate permeability observed after a 30-d-surgical denervation. Thirty days after a single systemic injection of colchicine the muscle phosphate metabolism is still perturbed especially in the slow soleus muscle in which the initial decrease in radioactive uptake associated with a 5-d-surgical denervation is inapparent. It is proposed that colchicine induces denervation-like alterations in the Pi- and POAS metabolism of skeletal muscles through mechanisms which are also activated -- partly or entirely -- by surgical denervation. These alterations do not result from the interruption of the axonal flow. A direct effect on the muscle membrane seems less likely than a disinhibition or activation of the muscle protein synthesis system.