Biosynthesis of Dolichol Phosphate Mannose in the Mitochondria1 Outer MembranePrevious studies have shown that mannose incorporation into mitochondrial glycoproteins is catalysed by a mannosyl-transferase, which is located in the mitochondrial inner membrane. It seems important to study the presence of mannosyl-transferases which are able to catalyse the transfer of mannose into other acceptors, such as polyprenic intermediates.Mouse liver mitochondria catalyse the incorporation of mannose from GDP-['4C]mannose into three products: most of the radioactivity is in a product (A) soluble in chloroform/methanol (2/1, by vol.) ; mannose is also incorporated into product (B) soluble in chloroform/methanol/water (10/10/3, by vol.), and into a product which is insoluble in organic solvents and trichloroacetic acid and which is a glycoprotein. We have tried to localize in mitochondria the mannosyl-transferases implicated in these reactions.Mitochondrial outer membranes were prepared by swelling purified mitochondria in phosphate buffer, and were purified on a discontinuous sucrose gradient. The purified outer membranes catalyse the incorporation of mannose from GDP-[14C]mannose in very large quantities into product A and in small quantities into product B.Different procedures (purification on silicic acid and DEAE-cellulose, hydrolytic procedures and chromatographic properties) demonstrated the identity of product A with authentic dolichol phosphate mannose.Product B is lipid-linked oligosaccharides. The biosynthesis of dolichol phosphate mannose depends on Mn" . When ethyleneglycol bis(2-aminoethyl)N,N'-tetracetic-acid (EGTA) is added to the mixture, the reaction is inhibited. When EGTA is added to a mixture containing preformed endogenous dolichol phosphate ['4C]mannose, the biosynthesis of the product stops, and the product is hydrolysed, with a concomitant apparition of lipid-linked-oligosaccharides. These results and the kinetics of the reactions suggest that dolichol phosphate mannose might be the mannosyl donor for the lipid-linked-oligosaccharides. The synthesis of dolichol phosphate mannose is reversed by the addition of GDP.The role of these products as intermediates in mitochondrial glycoprotein biosynthesis is discussed.Des travaux anterieurs ont montrk qu'une biosynthese autonome de glycoproteines est possible dans les mitochondries de foie ou de cerveau [1,2]. Plus specialement, le transfert de mannose, a partir de GDP-mannose, sur un accepteur proteinique endogene, est catalyse par une mannosyl-transferase locaAbbrlviutions. Mes, 2-(N-morpholino)ethane acide sulfonique; EGTA, ethyleneglycol bis(2-aminoethylj-N, N'-tetracetate.Enzymes. Monoamine oxydase (EC 1.4.3.4); cytochrorne c oxydase (EC 1.9.3.1); 5'-nucleotidase (EC 3.1.3.2); glucose-6-phosphatase (EC 3.1.3.9). lisee specifiquement dans la membrane mitochondriale interne, et absente de la membrane externe, de l'espace intermembranaire ou de la matrice [ 3 ] . La biosynthkse du noyau glycannique fondamental des glycoproteines fait intervenir des interm...

The relative value of each lactic dehydrogenase isoenzyme (iso LDH) was measured by electrophoretic separation in the serum and the pleural fluid of 100 patients. In each case, the cause of the pleural effusion was known. Two types of LDH isoenzyme pattern were found in the serum: a normal type with a low value of LDH 5 and an abnormal type with a high value of LDH 5. This high LDH‐5 level is due to an impaired liver function. In the pleural fluid, the electrophoretic patterns of five LDH isoenzymes were found by computerized processing. During congestive heart failure (28 cases) the electrophoretic pattern of the LDH isoenzymes was always similar in the serum and in the pleural fluid (transudative pleural effusion). During thoracic empyema, the relative values of the isoenzymes in the pleural fluid were regularly increasing from LDH 1 to LDH 5. In this situation, the evaluation of LDH 5 appeared to emanate from the increased granulocytes in the pleural fluid. In 22 inflammatory pleural effusions, the relative values of the five isoenzymes were equal. During malignant effusions (35 cases) a high level of LDH 5 was found in 21 patients. LDH 5 is known to be secreted by malignant tissue, and the authors confirmed it by finding a high level of LDH 5 in biopsy specimens of patients with mesothelioma or epidermoid lung cancer (7 cases). Conversely, the level of LDH 5 was low in biopsy specimens from normal lung tissue or benign inflammatory pleuritis (6 cases). Among the 14 patients with low levels of LDH 5 in the pleural fluid during malignant pleural effusion, the authors found the malignant lymphomas (three cases) and the small cell lung carcinoma (five cases). In these cases, the low level of LDH 5 was in agreement with the result of a low level of LDH 5 found in the biopsy of a metastatic liver localization of a small cell lung carcinoma. So the electrophoretic determination of LDH isoenzymes pattern in pleural fluid is a sensitive tool for the management of pleural effusion.

The cell wall compositions of two strains of "true" nocardiae, Nocardia asteroides R 399 and N. caviae IM 1381, and two strains of "so-called nocardiae," N . piracicabensis and N. mediterranei, were determined. Two different methods were used for preparing the cell walls. In the one, the bacteria were sonically treated, and the cell walls were obtained by differential centrifugation; in the other, the bacteria were delipidated before sonic treatment. The cell walls of true nocardiae contain nocardic acids, identified by comparison with nocardic acids isolated from the whole cell. The peptidoglycan structures of the "true" and "so-called" nocardiae studied showed some important differences. I n the true nocardiae, the glycan strand is constituted of P-N-acetylglucosaminyl ( 1 -+ 4) N-glycolylmuramic acid, and muramic acid is N-acetylated in the strains of "so-called nocardiae," as it is in the majority of bacteria. In addition, the peptide monomers of true nocardiae are diamidated on both the a-carboxyl group of wglutamic acid and the (L) carboxyl group of meso-diaminopimelic acid, whereas the peptide monomers of the "socalled nocardiae" have only one amide substituent on the carboxyl group of mesodiaminopimelic acid. I t is difficult to differentiate without ambiguity members of the genus Nocardia from the mass of the other actinomycetes which contain major amounts of meso-diaminopimelic acid. Our results suggest that true nocardiae may be those actinomycetes whose cell walls contain, in addition to arabinose and galactose, N-glycolylmuramic acid in the glycan part of the peptidoglycan and diamidated peptides in the peptide part of the peptidoglycan. In addition, true nocardiae contain nocardic acids.The differentiation between nocardiae and related organisms is often quite difficult. Morphologically the genus Nocardia contains filamentous, branching bacteria (19). The validity of this characteristic as a differential criterion has been criticized in past years, and biochemical or chemical tests have been proposed to establish a more precise classification (20). Since the initial work of Cummins (7, 8), cell wall composition has proven to be most useful in classifying organisms belonging to the order Actinomycetales. For example, LL-diaminopimelic acid (Dpm) is found in the cell walls of streptomycetes and rneso-Dpm in the cell walls of nocardiae (2).Moreover, some sugars, such as arabinose and galactose, are found in the celi walls of nocardiae but not in those of streptomycetes. Recently it has been proposed that the specific lipids present in whole cells of nocardiae be used to differentiate nocardiae from streptomycetes (27) and from mycobacteria (17,21). Since the first isolation of nocardic acids from Nocardia asteroides (25) and the determination of their structure (3, 4), nocardic acids with related structures have been found in all the species of Nocardia so far examined. Nocardic acids are a-branched, P-hydroxylated acids of the mycolic type; they have recently been found in the cell walls of Nocardia ...

Sixteen rabbits were killed 12 weeks after sectioning of the right knee anterior cruciate ligament. The left unoperated knee served as a control. The surface area of fibrillated cartilage from femoral condyles and tibial plateau was evaluated and expressed as a percentage of articular surfaces area. Cartilage from the femoro-patellar surfaces was homogenized for the quantification of several degradative activities, based on the release of digested products. Acid phosphatase, several glycosidases and neutral protease activity from the operated joint cartilage were significantly elevated, while collagenolytic activity was unmodified. The percentage of fibrillated cartilage correlated positively with arylsulfatase, glucosidase and neutral protease but negatively with mannosidase and fucosidase. The results may be consistent with the hypothesis of a sequential degradative process leading to cartilage destruction.