Immunohistochemical study of the adenohypophysis of the monkey Macaca irus with the use of antibodies anti 1-24-ACTH, anti 17-39-ACTH, anti alpha-and beta-endorphins, anti beta-LPM]

Indirect immunofluorescence technique with anti1-24- and anti17-39 ACTH, anti alpha- and anti beta-endorphins, anti beta-LPH sera has allowed us to detect a cellular type in the anterior lobe of the hypophysis of Macacus irus which react simultaneously with these five antisera. These cells are especially localized in the ventro-medial zone, but there are also present in the pars distalis, under the glandular capsule, and in the lateral lobes, amid the other cellular types. The cells of the intermediate lobe react on the whole with anti1-24-, these antisera are also immunoreactive with the anti alpha- and anti17-39ACTH and anti beta-LPH ; SOME CELLS, WHich react with anti beta-endorphin antisera. The adenohypophysis of Macacus irus contains therefore two categories of cells reacting with the above mentioned antisera : one of this type, localized in the anterior lobe and in the intermediate lobe, react simultaneously with the five antisera, the other type, localized only in the intermediate lobe does not react with the antiendorphins antisera.     

Immunocytological study on the differentiation of chick and quail adenohypophysis epithelial rudiments, grafted or cultivated in vitro: evidence for polypeptidic hormones

Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11--13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio-allantoic membrane exhibited cells reacting with the following immune sera: anti-beta-(1--24)ACTH, anti-alpha-(17--39)-ACTH, anti-alpha-endorphin, anti-beta-endorphin and anti-beta-LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to beta-(1--24)-ACTH, alpha-(17--39)-ACTH, and beta-LPH, as did adenohypophysis from quail embryos of corresponding age (9--10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder-cells and/or secrete some factors promoting differentiation.     

Comparative cellular localization of corticotropin and melanotropin in lerot adenohypophysis (Eliomys quercinus)

Summary Corticotropin and melanotropin producing cells were localized in the adenohypophysis of normal Lerots by using antibodies against synthetic corticotropins (anti 1–24 ACTH, anti 17–39 ACTH, anti 25–39 ACTH), and melanotropins (anti MSH, anti MSH). All the anticorticotropin sera stained the same cells both in the anterior lobe and in the intermediate lobe. The anti MSH serum only stained a few cells, exclusively located in the intermediate lobe. These MSH cells were not stained with anticorticotropin antibodies. The anti MSH serum revealed all the cells stained with anticorticotropin and anti MSH sera. Absorption tests showed that the 4–10 heptapeptide common to ACTH and MSH, is not responsible for the immunohistochemical staining. The staining of only some corticotrophs with the anti 4–10 ACTH serum might indicate the presence in these cells of a peptide with an accessible 4–10 site. These results are discussedWe thank A. Pillez for technical assistance (C.N.R.S.). This work was supported by a grant from U.E.R. III Lille 1976Attaché de Recherche INSERM     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

Synthesis of C-glycopyranosyl compounds by a palladium-catalyzed coupling reaction of 1-tributylstannyl-D-glucals with organic halides.

1-Tributylstannyl-D-glucals, prepared from the corresponding 1-phenylsulfonyl-D-glucals, were coupled efficiently to various organic halides in the presence of a palladium(0) catalyst. This mild reaction is specially useful for the preparation of 1-C-aryl-D-glucals and compatible with unprotected hydroxy groups or hindered aromatic bromides. It has been shown that the resulting 1-C-aryl(alkyl)-D-glucals are suited for further synthetic manipulation of the enol ether group, including stereoselective hydrogenation, hydroboration-oxidation, or epoxidation. All compounds formed resulted from the attack of the alpha-face of the glucal derivatives by the reagent. The reaction, extended to 1,3-, 1,4-di-, and 1,3,5-tri-bromobenzenes, leads to the corresponding symmetrical di-(tri)-C-glucosylbenzenes. Finally, a sequential di-C-glucosylation of 1,3-dibromobenzene with two different 1-stannylated glucals was obtained.     

Vitamin K dependent carboxylation: determination of the stereochemical course using 4-fluoroglutamyl-containing substrate

The stereochemical course of the vitamin K dependent carboxylation has been elucidated using a (4S)-4-fluoroglutamyl-containing pentapeptide as a substrate. The absolute configuration of the [13C]-4-carboxy-4-fluoroglutamate obtained when the carboxylation was carried out with 13C-labeled sodium bicarbonate, was determined after reduction of the [13C]-4-carboxy-4-fluoroglutamyl residue into 4-fluoro-5,5'-dihydroxyleucine, hydrolysis, lactonization, and peracetylation. The absolute configuration at C-4 was determined to be S by locating the 13C label in the lactone ring of the trans isomeric lactone and in the hydroxymethyl group of the cis isomer following HPLC separation of both isomers and analysis by GC/MS/MS techniques. It follows that the vitamin K dependent carboxylation occurs with inversion of configuration.     

Phospholipid metabolism modulates cyclic nucleotide phosphodiesterase activity in rat heart microsomes

Cyclic nucleotide phosphodiesterase activity in rat heart microsomes is attributable to several isoenzymatic forms: a cyclic AMP-specific, a cyclic GMP-specific, and a cyclic GMP-stimulated enzyme. Incubation of microsomes with an exogenous phospholipase C (C. welchii) induced a marked stimulation (+126%) of cyclic AMP phosphodiesterase and a moderate stimulation (+49%) of cyclic GMP-phosphodiesterase in the membrane-bound fraction. Besides, a notable fraction of activity was solubilized by the treatment. A parallel decrease in the activating effect of cyclic GMP on the hydrolysis of cyclic AMP was observed in the membranes (down to 18% of the control effect). It resulted from a marked stimulation of the basal activity, while the activated level was unaffected. The treatment by an exogenous phospholipase D induced more moderate modifications. The addition to microsomes of oleyl,acetyl-glycerol, but not of long chain-diacylglycerols, partly reproduced the phospholipase C effect. Phosphatidate also induced variations in phosphodiesterase activity, and could thus participate in the phospholipase effects. These results suggest that endogenous phospholipases, the activity of which is modulated by hormonal stimuli, might influence phosphodiesterase activity in cardiac membranes by producing phospholipid metabolites, with potential consequences on heart contractility.     

Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion [Lederer, F., & Mathews, F.S. (1987) in Flavins and Flavoproteins, Proceedings of the Ninth International Symposium, Atlanta, GA, 1987 (Edmondson, D.E., & McCormick, D.B., Eds.) pp 133-142, Walter de Gruyter, Berlin]. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)