[Diagnosis of pulmonary tuberculosis: evaluation of the TB IgA EIA assay in Morocco]. / Diagnostic de la tuberculose pulmonaire: evaluation du test "TB IgA EIA" au Maroc.

We analysed 2 evaluation lots of the TB IgA EIA test in pulmonary tuberculosis patients (TBp). Sera were obtained from 345 TBp, 18 healthy subjects (HS), 28 subjects in contact with tuberculous patients (CS) and 16 non-tuberculous lung disease patients (N-TB) for the first evaluation lots and 302 TBp, 60 HS, 21 CS and 18 N-TB for the second. IgA titres against p-90 antigen with the second evaluation lot were significantly higher than the first evaluation lot. With the second evaluation lots, the sensitivity was 78.8% whereas with the first evaluation lot, the sensitivity was 75.9%. Specificity for the first and second evaluation lots was 50% and 70.7% respectively. The sensitivity of this test is still not satisfactory to establish pulmonary tuberculosis diagnosis.

[Diagnosis of pulmonary tuberculosis: evaluation of the TB IgA EIA assay in Morroco]

We analysed 2 evaluation lots of the TB IgA EIA test in pulmonary tuberculosis patients [TBp]. Sera were obtained from 345 TBp, 18 healthy subjects [HS], 28 subjects in contact with tuberculous patients [CS] and 16 non-tuberculous lung disease patients [N-TB] for the first evaluation lots and 302 TBp, 60 HS, 21 CS and 18 N-TB for the second. IgA titres against p-90 antigen with the second evaluation lot were significantly higher than the first evaluation lot. With the second evaluation lots, the sensitivity was 78.8% whereas with the first evaluation lot, the sensitivity was 75.9%. Specificity for the first and second evaluation lots was 50% and 70.7% respectively. The sensitivity of this test is still not satisfactory to establish pulmonary tuberculosis diagnosis

[Apolipoprotein E and angiotensin-converting enzyme gene polymorphisms as risk factors of coronary disease]. / Implication du polymorphisme génétique de l'apolipoprotéine E et de l'enzyme de converstion de l'angiotensine dans l'athérosclérose coronarienne.

The objective of this study was to test the hypothesis that apo E (RFLP, HhaI) and/or angiotensin-converting enzyme (ACE) (ins16del) are associated with higher risk for coronary heart disease. We investigated 250 patients who underwent complete cardiac examination comprising coronary angioplasty and biological analysis (CT, HDLc, LDLc, TG, apo A and apo B). Prevalence of the alleles of apo E and ACE was assessed by molecular analysis. Patients without stenosis or with non-significant stenosis (> 50% of the vascular lumen) were used as reference group (141 patients). Those presenting a significant stenosis of the coronary artery (. 50% of the vascular lumen) were considered as cases (109 patients). The relative frequency of the e 4 allele was significantly higher in cases than in reference group (p > 0.02). A strong association have been found between coronary heart disease and apo E polymorphism (2 = 8.91; p > 0.05). The presence of the e 4 allele increase the risk of atherosclerosis (RR = 2.71; IC95%: 1.25-5.90; p > 0.02) compared to e 3 allele. Also, subjects with D allele were more frequent in cases than in reference group (p > 0.001). A significant association was noted between ACE polymorphism and coronary heart disease (2 = 42.15; p > 0.001). This relationship was positive (rho de Spearman = 0.39; p > 0.01). With D/D homozygotes patients, the RR for coronary heart disease was 19.10 (p > 0.001), while The RR with I/D heterozygotes was 6.91 (p > 0.001) compared to I/I homozygotes. A significant interaction have been shown up between D/D genotype and arterial hypertension (HTA) (2 de Wald = 16.10; p > 0.001). The multivariate analysis showed that the chronic smoking, diabetes, hypoapolipoproteinemia A, interactive effects between D/D and HTA, I/D and obesity, and between D/D and hypertriglyceridemia were the major significant factors to take into consideration in our population. We also note that subjects with both D and e 4 alleles were presenting a high risk to coronary heart disease (RR = 5.93; IC95%: 2.00-17.55; p > 0.01). Thus, those two alleles (4 and D) appears to be important cardiovascular risk factors in the moroccan population.

[Homocysteine, lipoprotein (a): risk factors for coronary heart disease]. / Homocystéine, lipoprotéine (a): acteurs de risque de l'athérosclérose coronarienne.

Our data suggest that the hyperhomocysteinemia and/or increased plasma level of lipoprotein Lp(a) are risk factors for coronary heart disease. We investigated 178 patients who underwent complete cardiac examination comprising coronary angiography and biological analysis (CT, HDL-c, LDL-c, TG, and apoAI, apoB, homocysteine and Lp(a)). Patients presenting a significant stenosis of the coronary artery ( 50% of the vascular lumen) were considered as cases (113 patients). Those without stenosis or with non-significant stenosis (< 50% of the vascular lumen) were used as controls (65 subjects). Homocysteinemia was significantly higher in cases than in control subjects (8.26 mol/L (2.34 versus 17.85 (2.34, p < 0.001). A strong association between coronary heart disease and homocystein has been found (Eta(2) = 0.76). The OR were 0.16 when homocystein level was lower than 15 mol/L, and 27.78 when homocysteine level was upper than or equal to 15 mol/L. The RR was 5.16 (95% IC = 3.66-6.66, p < 0.001). Even though there was a significant correlation between tabagic impregnation and homocysteinemia (Spermann's rho = 0.37, p < 0.05), there was no interactive effect between these two factors and coronary disease (Wald khi2 = 0.086, p > 0.05). Therefore, no association was found between homocyteinemia and other coronary heart disease risk factors. The Lp(a) levels were significantly higher in cases than in controls subjects (188 (84 mg/L in control subjects versus 590 (199 in cases, p < 0.001). A stronger relationship was noted between coronary heart disease and Lp(a) (Eta (2) = 0.66). The OR were 0.09 when lipoprotein (a) levels were lower than 350 mg/L, and 5,88 when Lp(a) levels were higher than or equal to 350 mg/L. The estimate RR was 6.47 (95% IC = 4.39-8.55, p < 0.001). The level of Lp(a) was positively correlated with the severity of coronary heart disease (Spermann's rho = 0.95, p < 0.001). A weak correlation between Lp(a) and LDL-c was observed (Spermann's rho = 0.12, p = 0.048). But the multivariate analysis didn't show interactive effect between these two factors and coronary disease (khi2 de Wald = 0.264, p > 0.05). No association was noted between Lp(a) and the others risk factors. Moreover, a positive correlation between the levels of homocysteine and those of Lp(a) was found (Spermann's rho = 0.54, p < 0.001). In contrast their effect on coronary heart disease seems to be independant (Wald khi2 = 2.957, p > 0.05). Thus, these two parameters appear as independant risk factors for coronary heart disease.

Inhibition of glycosidases by Astragalus lusitanicus and correlation with toxicity.

This study determined whether Astragalus lusitanicus inhibits glycosidase enzymes other than alpha-mannosidase. Plasma collected from lambs given fresh A lusitanicus inhibited beta-glucosidase and beta-galactosidase, indicating the presence of inhibitors in their blood. The residual activity of these enzymes was also modified in tissues of dead animals. beta-glucosidase activity was reduced in liver and kidney specimens with pronounced effects in tissues of animal that presented with prominent clinical signs of poisoning; beta-galactosidase activity was decreased by 88.5 to 95% in kidney, while that of liver remained unchanged. Fractions of the plant butanol extract inhibited the gycosidase enzymes. Chemical analysis revealed the presence of hypaphorin in the extract of A lusitanicus. As a tryptophan derivative, this alkaloid may play a role in the toxicity of this legume.

Location of the cytoplasmic epitope for a K(+)-competitive antibody of the (H+,K+)-ATPase.

The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity. To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cys529 and Glu561. This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M. The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M. The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface. The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited. The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites.

Immunopurification of gastric parietal cell tubulovesicles.

1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.

H+/K(+)-ATPase contents of human, rabbit, hog and rat gastric mucosa.

A monoclonal antibody (mAb 95-111) was used to titrate the amounts of H+/K(+)-ATPase in subcellular fractions of the fundus of rats, pigs, rabbits and humans. All four had similar amounts of H+/K(+)-ATPase: 2.1 +/- 0.5 (human), 1.9 +/- 0.4 (rabbit), 4.4 +/- 0.5 (rat) and 4.2 +/- 0.8 (hog) mg ATPase/g wet tissue. The antigen concentrations and H+/K(+)-ATPase enzymatic activities of subcellular fractions were linearly correlated in all species but rat suggesting that human, rabbit and hog H+/K(+)-ATPases have similar rates of catalysis (223 mumol Pi/h per mg ATPase). The non-correlation of rat data probably reflects the known lability of the rat enzyme. Hence, immuno-titration promises to be a more reliable method of estimating rat ATPase than the currently used enzymatic assay. The H+/K(+)-ATPase content of human biopsies was 20-fold higher than previously-published (Smolka, A. and Weinstein, W.M. (1986) Gastroenterology 90, 532-539) suggesting that the previous immuno-titration underestimated the H+/K(+)-ATPase content of the human fundus.

A monoclonal antibody which inhibits H+/K(+)-ATPase activity but not chloride conductance.

A mouse monoclonal antibody was raised against hog gastric membranes. This antibody (95-111 mAb) has a very high affinity for the 95 kDalton band of H+/K(+)-ATPase-enriched membranes, and does not react with Na+/K(+)-ATPase. The epitope is located on the tubulovesicles and canaliculi of the parietal cells. The 95-111 mAb also inhibits the ATP hydrolytic activity, decreases the steady-state phosphorylation level and inhibits the phosphatase activity of H+/K(+)-ATPase, strongly suggesting that the epitope is on the catalytic subunit of H+/K(+)-ATPase. The 95-111 mAb also recognizes rat, rabbit and human gastric H+/K(+)-ATPase. This mAb differs from the H+/K(+)-ATPase-inhibiting mAb previously described (Asano et al. (1987) J. Biol. Chem. 262, 13263-13268), in that it does not inhibit the chloride conductance opened by Cu-o-phenanthroline in gastric vesicles.

Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1.

The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.

The ontogeny of rat gastric H+/K+-ATPase.

The ontogeny of rat H+/K+-ATPase was studied between foetal day 18 and neonatal day 18, using a specific monoclonal antibody (95-111 mAb). The H+/K+-ATPase content of gastric subcellular membranes was assayed and the ATPase subunits were characterized by Western blot. The epithelium density in parietal cells was measured by immunohistochemistry. H+/K+-ATPase was present in the 18-day-old foetuses and parietal cells were detected on foetal day 19. The H+/K+-ATPase concentration remained stable from foetal day 18 to neonatal day 1, while the parietal cell density increased 2.5-fold. The H+/K+-ATPase concentration increased by 2.5-fold on day 6, then remained constant up to day 18. The parietal cell density remained unchanged during this period, suggesting that the concentration increase on day 6 was due to an increase in parietal cell ATPase content. The 95-111 mAb recognized a 95 kDa single band on foetal day 18 and a doublet at all the other stages of development. Previous studies had demonstrated that acid secretion drops critically at day 12 post partum in the rat and that H+/K+-ATPase activity is lost. The present study demonstrates that the H+/K+-ATPase is, however, present on day 12.

[Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin]. / Purification jusqu'à apparente homogénéité du récepteur de la somatostatine de la lignée cellulaire humaine HGT-1 d'origine gastrique.

This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.

Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa.

The presence in human gastric juice of a lipase secreted by the gastric mucosae has been reported previously, but its exact cellular origin has not yet been established. Polyclonal antibodies specific to human gastric lipase (HGL) were prepared, and used by an immunofluorescence technique to label cells producing HGL. This immunocytolocalization was correlated with that of pepsin (chief cells) and parietal cells using specific polyclonal or monoclonal antibodies. Our results clearly establish that HGL is exclusively located in the chief cells of fundic mucosa; furthermore, it was found to be always co-located with pepsin. No HGL was observed in the parietal or mucus cells. HGL was always detected intracellularly, either in secretory granules of the apical region of the chief cells, or revealed by more diffuse cytoplasmic labelling.

Thiophosphorylation of hog gastric (H+ + K+)-ATPase membranes by endogenous protein kinases.

(H+ + K+)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [gamma-32P]ATP or [gamma-35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg2+ and 5 microM [gamma-32P]ATP, rapid and transient incorporation of 32P occurred at 0 degrees C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H+ + K+)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0 degrees C, and in the presence of 10 mM manganese and 5 microM ATP[S], 0.58 +/- 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36-40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase-dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682-11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator.

Porcine pancreatic lipase. The disulfide bridges and the sulfhydryl groups.

Following complete sequence analysis of the 449 amino acids in porcine pancreatic lipase [J. De Caro et al. (1981) Biochim. Biophys. Acta, 671, 129-138], the position of the six disulfide bridges and of the two free thiols of the protein was investigated using a variety of techniques. Three bridges (Cys-4--Cys-10, Cys-237--Cys-261 and Cys-433--Cys-449) were easily identified in the peptic digest of lipase at pH 2.0. In the latter digest, two other bridges (Cys-285--Cys-296 and Cys-299--Cys-304) were also identified by means of the cystine peptide constituted by two peptide segments: Ala281-Gly-Phe-Pro-Cys-Asp-Ser287 and Thr292-Ala-Asn-Lys-Cys-Phe-Pro-Cys-Pro-Ser-Glu-Gly-Cys-Pro-Gln-Met307. A disulfide bridge, formed by Cys-285 and one of the three half-cystines of the other segment, connected the two peptide moieties. A second disulfide bridge linked the two remaining half-cystines. It was not possible to split any peptide bond between Cys-296 and Cys-304 with proteolytic enzymes. The determination of the pairing of the four half-cystines was resolved as follows. Bond Lys-295--Cys-296 was cleaved with trypsin. A single cycle of Edman degradation was then performed on the peptide compound, thus freeing, in particular, Cys-296 of the peptide bond (296-297). Cys-296 only retained its S-S connection with the half-cystine partner. At the completion of the above operations, the two peptide segments (282-287) and (297-307) could be separated. Consequently it was concluded that Cys-285 is linked to Cys-296. Therefore Cys-299 and Cys-304 are paired. The most reactive SHI group of the enzyme was localized on Cys-181 by condensation of the native protein with radioactive N-ethylmaleimide. The alkylation of the SHII group required previous denaturation of the molecule at alkaline pH. The results obtained for the characterization of the SHII group suggested the existence of two isomeric forms, the SHII being either on Cys-101 or Cys-103 and the bridge alternately between Cys-90--Cys-103 or Cys-90--Cys-101. It is not yet known whether these two forms pre-exist in native lipase or result from an exchange reaction. The bridge Cys-90--Cys-101 was characterized in a thermolytic digest of a cyanogen bromide fragment (CN II) of the protein. However, another bridge involving Cys-181 was also found in the digest. This bridge is considered as being an artefact. It is possible that considering the treatments undergone by the large peptide (CN II), the SH groups of lipase were oxidized and transformed in an S-S bridge. The disulfide bridges of lipase form relatively small loops along the main chain. This arrangement is consistent with a high flexibility of the molecule. As reported earlier [R. Verger et. al. (1971) Biochim. Biophys. Acta. 242, 580-592], the SHI group is not essential for lipase activity. The role of the SHII group should be more precisely investigated.

Characterization of the serine reacting with diethyl p-nitrophenyl phosphate in porcine pancreatic lipase.

The position in porcine pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) of the serine reacting specifically with emulsified or micellar diethyl p-nitrophenyl phosphate has been investigated. This serine which appears to be involved in lipase adsorption to insoluble triglyceride interfaces, is at position 152 in the enzyme chain. The sequence around this amino acid is: His-Val-Ile-Gly-His-Ser-Leu-Gly.