Screening of preduodenal lipases in several mammals.

The tissular localization of preduodenal lipases was studied from the tongue to the pyloric portion of the stomach in 11 mammals. Lipolytic activities were clearly differentiated from those of pancreas. All lipase activities show an acidic pH optimum, except the gastric enzyme from hog. For every mammal tested, preduodenal lipase activity was associated mainly with only a single tissue located either in tongue, or in the pharyngeal area, or in the stomach. Resistance to acidic pH medium allows the classification of lipase activities into three groups. These results are related to the dietary habits and zoologic classification of the different animal species.

Purification, characterization and kinetic properties of the rabbit gastric lipase.

Rabbit gastric lipase was purified from an acetonic powder of rabbit stomach fundus. 25 mg of pure rabbit gastric lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained from 30 rabbit stomachs after ammonium sulfate fractionation, Sephadex G-100 gel filtration and cation exchange (mono S column) using a fast protein liquid chromatography (FPLC) system. The pure enzyme obtained was resistant to acidic pH conditions, and had specific activities of 1200, 850 and 280 U/mg, using, respectively, short- (tributyroylglycerol (TC4)), medium- (trioctanoyl- to tridecanoylglycerol (TC8-TC10)) and long-chain (soybean oil) triacylglycerols. The amino-acid composition was determined, and the first 30 N-terminal amino-acid residues were sequenced. Interfacial denaturation and catalytic properties on triacylglycerol emulsions were studied. Rabbit gastric lipase turned out to be structurally and kinetically very similar to human gastric lipase.

Inactivation of pancreatic and gastric lipases by THL and C12:0-TNB: a kinetic study with emulsified tributyrin.

THL is a potent inhibitor of pancreatic (PPL) and gastric (HGL, RGL) lipases. Inactivation occurs preferentially at the oil/water interface (method B, C). In the aqueous phase (method A), the inhibition of HGL was accelerated by the presence of bile salts. C12:0-TNB, a disulfide reagent, specifically inactivates gastric lipases and had no effect on the pancreatic lipase (in the presence of bile salts) whatever the method used. The capacity of THL and C12:0-TNB to inactivate lipases using Methods B and C was found to depend directly upon the interfacial area of the system used. Consequently, inactivation can be reduced or prevented by further addition of a water-insoluble substrate which reduces the surface density of inactivator molecules. With a heterogeneous system of this kind, typical of lipolysis, the use of a classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (Km, KI, Vmax) are only apparent values.

Ajoene prevents fat digestion by human gastric lipase in vitro.

Human gastric lipase (HGL) is a sulfhydryl enzyme which has been shown by Gargouri et al. (Gargouri, Y., Moreau, H., Piéroni, G. and Verger, R. (1988) J. Biol. Chem. 263, 2159-2162) to be inhibited by hydrophobic disulfides. Since HGL is involved in the digestion and absorption of dietary fats we have investigated in vitro the ability of ajoene, a natural disulfide to inactivate HGL. Ajoene is derived from ethanolic garlic extracts. The finding that ajoene inactivates HGL is consistent with the fact that it is reactive towards sulfhydryl compounds and also corroborates previous reports on the ability of garlic to lower triacylglycerol blood levels. These data may explain the age-old Mediterranean and Oriental belief in the 'blood-thinning' effects of garlic on a molecular and physiological basis.

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

BACKGROUND: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance. METHODS AND RESULTS: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The PEGylated variants recognized only one third of the antibodies elicited with wild-type SakSTAR in AMI patients. In experimental animals, plasma clearances were reduced 2. 5- to 5-fold with P5, 5- to 20-fold with P10, and 20-fold with P20, and bolus injection induced pulmonary plasma clot lysis at doses inversely related to their clearance. Intravenous bolus injection of 5 mg of the P5, P10, or P20 variants in AMI patients was associated with plasma half-lives (t(1/2alpha)) of 13, 30, and 120 minutes and clearances of 75, 43, and 8 mL/min, respectively, compared with 3 minutes and 360 mL/min for SakSTAR. Injection of 5 mg P5 variant restored TIMI-3 flow within 60 minutes in 14 of 18 AMI patients (78%, 95% CI 55% to 91%) and of 2.5 mg in 7 of 11 patients (63%, 95% CI 35% to 85%), both in the absence of fibrinogen degradation. The immunogenicity of the variants was significantly (P:<0.002) reduced. CONCLUSIONS: The staphylokinase variant SY161-P5, derivatized with one linear polyethylene glycol molecule of M:(r) 5000, is a promising fibrin-selective agent for single-bolus coronary thrombolysis.

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.

The first green lineage cdc25 dual-specificity phosphatase.

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.

Interplay between the genetic clades of Micromonas and their viruses in the Western English Channel.

The genus Micromonas comprises distinct genetic clades that commonly dominate eukaryotic phytoplankton community from polar to tropical waters. This phytoplankter is also recurrently infected by abundant and genetically diverse prasinoviruses. Here we report on the interplay between prasinoviruses and Micromonas with regard to the genetic diversity of this host. For 1 year, we monitored the abundance of three clades of Micromonas and their viruses in the Western English Channel, both in the environment using clade-specific probes and flow cytometry, and in the laboratory using clonal strains of Micromonas clades to assay for their viruses by plaque-forming units. We showed that the seasonal fluctuations of Micromonas clades were closely mirrored by the abundance of their corresponding viruses, indicating that the members of Micromonas genus are susceptible to viral infection, regardless of their genetic affiliation. The characterization of 45 viral isolates revealed that Micromonas clades are attacked by specific virus populations, which exhibit distinctive clade specificity, life strategies and genetic diversity. However, some viruses can also cross-infect different host clades, suggesting a mechanism of horizontal gene transfer within the Micromonas genus. This study provides novel insights into the impact of viral infection for the ecology and evolution of the prominent phytoplankter Micromonas.

Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands.

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of rabbit gastric lipase and pepsinogen.

Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.

Importance of sulfhydryl group for rabbit gastric lipase activity.

We have shown recently that rabbit gastric lipase (RGL) purified from gastric tissue presents catalytic properties comparable with those of human gastric lipase (HGL). We report here that only one sulfhydryl group was modified per molecule of native RGL after incubation at pH 8.0 with 5,5'-dithiobis(2-nitrobenzoic acid) (NbS2) for 4 h or 4,4'-dithiopyridine (4-PDS) for 60 min. With both reagents, a direct correlation was found between the modification of one sulfhydryl group per enzyme molecule and loss of RGL activity. Incubation of RGL with the new hydrophobic sulfhydryl reagent, dodecyldithio-5-(2-nitrobenzoic acid) (C12-NbS), at 30-fold molar excess, at pH 3.0, 5.0 and 8.0, induced immediate and complete inactivation of RGL. Unlike NbS2 and 4-PDS, C12-NbS almost instantaneously stopped the course of tributyrin hydrolysis by RGL, in contrast to porcine pancreatic lipase (PPL). RGL can be included with HGL in the group of sulfhydryl enzymes.

mCD24 expression in the developing mouse brain and in zones of secondary neurogenesis in the adult.

Interactions mediated by cell surface glycoproteins are considered to be crucial during the formation of the nervous system. Using a monoclonal antibody directed to mCD24, a glycosylphos-phatidylinositol-anchored membrane glycoprotein, we have mapped its distribution throughout the mouse cerebral cortex during development and in young adult. Before birth, mCD24 immunoreactivity was observed in the intermediate zone, the cortical plate and the marginal zone, whereas the ventricular zones were immunonegative. After birth, mCD24 expression declined rapidly in the cortex, except in the corpus callosum (and other commissures in the brain) where immunoreactivity was still found until P20. Furthermore, mCD24 expression was maintained in young adults (until P60, at least) in zones of secondary neurogenesis, such as the granule cells of the dentate gyrus, the subventricular zone lining the anterior part of the lateral ventricles and a zone of cells extending between the striatum and the corpus callosum to the centre of the olfactory bulb. In this area mCD24 and polysialic acid neural cell adhesion molecule stainings were superimposed, and this corresponded to the pathway of migration of the olfactory immature neurons (subependymal layer). A layer of ciliated ependymal cells, lining all the ventricular walls, was also immunoreactive for mCD24. Thus, except for these epithelial-like cells, mCD24 was essentially found associated with differentiating postmitotic neurons. Its spatiotemporal expression, both during development and in the adult, is compatible with a role for this glycoprotein in cell surface recognition and in signalling events occurring during neuronal migration and axonal growth.

Procoagulant properties of intravenous staphylokinase versus tissue-type plasminogen activator.

The fibrin-specificity and procoagulant effects of recombinant staphylokinase (Sak42D) were compared with those of recombinant tissue-type plasminogen activator (rt-PA) in patients with acute myocardial infarction. Plasma samples were obtained at baseline and at 25 and 90 min, from 24 patients who were randomly assigned to a double bolus (15 mg each, 30 min apart) administration of Sak42D or to accelerated weight-adjusted rt-PA (maximum of 100 mg over 90 min). Baseline levels of fibrinopeptide A (FPA), prothrombin fragment 1 + 2 and thrombin-antithrombin III complex (TAT) were comparable in the Sak42D and rt-PA groups (p > or = 0.6). In patients treated with Sak42D, plasma levels of FPA, prothrombin fragment 1 + 2 and TAT did not markedly increase during treatment (p = 0.06, p = 0.4 and p = 0.03, respectively). In contrast, during administration of rt-PA the levels of FPA, prothrombin fragment 1 + 2 and TAT increased significantly over baseline (p = 0.003, p < 0.0001 and p = 0.001, respectively). As a result, the levels of all three procoagulant parameters were significantly lower during treatment with Sak42D as compared to rt-PA. Thus, FPA levels in the Sak42D group (median values) were 40 ng/ml at 25 min and 11 ng/ml at 90 min, as compared to 88 ng/ml and 50 ng/ml in the rt-PA group (p = 0.0007 and p = 0.009, respectively). Prothrombin fragment 1 + 2 levels in the Sak42D group were 1.3 nM at 25 min and 1.2 nM at 20 min, as compared to 11 nM and 5.3 nM in the rt-PA group (both p < 0.0001). TAT levels were 4.7 ng/ml at 25 min and 6.2 ng/ml at 90 min in the Sak42D group, with corresponding values of 16 ng/ml and 9.6 ng/ml in the rt-PA group (p = 0.02 and p = 0.03, respectively). In the patients treated with Sak42D, no significant systemic fibrinolytic activation was observed, as revealed by unaltered levels of clottable fibrinogen, plasminogen and alpha 2-antiplasmin up to 90 min after the start of therapy. In contrast, the corresponding residual levels at 90 min in patients treated with rt-PA decreased to (mean +/- SEM; n = 12) 62 +/- 6%, 45 +/- 5% and 52 +/- 10%, respectively (all p < or = 0.01 versus the Sak42D group). These data confirm the high degree of fibrin-specificity of Sak42D and demonstrate that this is associated with significantly less generation of procoagulant activity in plasma after intravenous administration in patients with acute myocardial infarction.

Prevalence and induction of circulating antibodies against recombinant staphylokinase.

Streptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients. Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 micrograms/ml (median 11 micrograms/ml) anti-STAR antibodies and 0.9 to 370 micrograms/ml (median 18 micrograms/ml) anti-SK antibodies (p < 0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 micrograms/ml (median 7.1 micrograms/ml) and 0.4 to 120 micrograms/ml (median 7.3 micrograms/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 micrograms/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 micrograms STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 micrograms/ml at baseline, 2.9 micrograms/ml at 6 to 8 days and 1.2 mg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 micrograms/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 micrograms/ml at baseline to 360 micrograms/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Multicenter evaluation of commercially available methods for the immunological determination of plasminogen activator inhibitor-1 (PAI-1).

In order to evaluate the comparability of data obtained with various available kits for the immunological determination of PAI-1 antigen in plasma and in order to investigate the underlying cause of observed differences, e.g. problems of specificity or of proper calibration of the provided standard, a multicenter study was organised in the framework of the Subcommittee of Fibrinolysis of the Scientific and Standardization Committee. Eight different plasma samples were distributed among 16 laboratories: a pooled normal plasma, NIBSC 87/512, PAI-1 antigen depleted plasma, PAI-1 depleted plasma supplemented with 59 ng/ml active PAI-1 and four different individual plasma samples. A considerable variation in absolute values is observed between the various kits, e.g. in pooled normal plasma a value is found ranging between 7.4 and 28 ng/ml. Harmonization of all data relative to the PAI-1-depleted plasma supplemented with an exact amount of active PAI-1 (59 ng/ml), followed by a statistical analysis using a two way analysis of variance, revealed that 6 out of 7 kits yielded values that were not significantly different with coefficients of variation around 30%. Correlations between the values obtained with these kits yielded slopes between 0.75 and 1.44 with correlation coefficients between 0.973 and 0.999. Values obtained with one kit appeared to be significantly different (even after harmonization) from the other kits (p < 0.001 to p < 0.05). Comparison of PAI-1 antigen with the PAI activity values in the analysed samples suggests that one kit may deal with a problem of a difference in reactivity between active and latent PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of nuclear WW domains and proline-rich proteins in dinoflagellate transcription.

Dinoflagellates are unique among eukaryotes in their lack of histones and nucleosomes, and permanently condensed chromosomes. These unusual features raise questions as how chromatin condensation and gene expression are achieved. In this study, we investigated nuclear proteins potentially implicated in the regulation of the transcription. Dinap1 is a dinoflagellate nuclear protein that has a WW domain and is synthesized mainly in G1 and S phases of the cell cycle. In this study, we found that Dip1, a proline-rich potential ligand of Dinap1, and DapC, a Dip1 potential ligand, were both present in the nucleus of Crypthecodinium cohnii during the G1 phase. Dip1 contained a PPXY motif, and its domain organization was similar to that of the splicing factor FBP21 in that it possessed one zinc finger and two WW domains. Although DapC has no known homolog, 22 repeats of a PPXPXGX heptapeptide were identified at the N-terminus, and this structure is similar to that of the C-terminal part of the mouse splicing factor SAP62. Dinap1 was co-precipitated with Dip1 and DapC in vitro and in vivo, but despite their nuclear location, these three proteins did not bind directly to DNA. Dinap1 activated up to 40% of the basal transcription activity of C. cohnii in an in vitro assay, whereas DapC inhibited it by 40% and Dip1 had no effect. These dinoflagellate proteins appear to be the subunits of a nuclear complex that may be involved in regulating transcription.

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.

Allergic enterocolitis presenting as recurrent necrotizing enterocolitis in preterm neonates.

An uncommon clinical entity mimicking necrotizing enterocolitis (NEC) is allergic enterocolitis secondary to cow's milk protein allergy. Although milk protein allergy is the most common food allergy among infants and young children, the incidence and prevalence of this disease entity presenting as enterocolitis in neonates is not well documented. We report this case of milk protein-associated allergic enterocolitis to highlight the unusual recurrent presentation as NEC, (with recurrent pneumatosis, bloody stools) managed successfully with modification of milk formula.