Red Flags for early referral of people with symptoms suggestive of narcolepsy: a report from a national multidisciplinary panel.

OBJECTIVE: Narcolepsy is a lifelong disease, manifesting with excessive daytime sleepiness and cataplexy, arising between childhood and young adulthood. The diagnosis is typically made after a long delay that burdens the disease severity. The aim of the project, promoted by the "Associazione Italiana Narcolettici e Ipersonni" is to develop Red Flags to detect symptoms for early referral, targeting non-sleep medicine specialists, general practitioners, and pediatricians. MATERIALS AND METHODS: A multidisciplinary panel, including patients, public institutions, and representatives of national scientific societies of specialties possibly involved in the diagnostic process of suspected narcolepsy, was convened. The project was accomplished in three phases. Phase 1: Sleep experts shaped clinical pictures of narcolepsy in pediatric and adult patients. On the basis of these pictures, Red Flags were drafted. Phase 2: Representatives of the scientific societies and patients filled in a form to identify barriers to the diagnosis of narcolepsy. Phase 3: The panel produced suggestions for the implementation of Red Flags. RESULTS: Red Flags were produced representing three clinical pictures of narcolepsy in pediatric patients ((1) usual sleep symptoms, (2) unusual sleep symptoms, (3) endocrinological signs) and two in adult patients ((1) usual sleep symptoms, (2) unusual sleep symptoms). Inadequate knowledge of symptoms at onset by medical doctors turned out to be the main reported barrier to diagnosis. CONCLUSIONS: This report will hopefully enhance knowledge and awareness of narcolepsy among non-specialists in sleep medicine in order to reduce the diagnostic delay that burdens patients in Italy. Similar initiatives could be promoted across Europe.

7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 µg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

Intestinal phospholipase, a novel enzyme.

We evaluated phospholipase activity in the intestine of rats and other species. Phospholipase activity was assayed by a surface barostat technique or an egg yolk titration system. Mucosal activity was found only by the surface barostat technique with phosphatidylglycerol as substrate; it was not found with phosphatidylcholine as substrate in assays by either technique. In gut luminal fluid activity was found when both phosphatidylcholine and phosphatidylglycerol were used as substrate in assays by the surface barostat technique, and phosphatidylcholine as substrate yielded activity in egg yolk titration. In rats in which pancreatic juice had been diverted, mucosal and gut luminal phospholipase activity was greater than in controls, thus demonstrating that enzyme activity was not due to pancreatic phospholipase. Bacterial origin of phospholipase activity was excluded in that phospholipase activity was found in germ-free rats; gastric and salivary gland origins were excluded in that continued phospholipase activity was found in rats with gastric fistula. The physiological importance of the enzyme was established by the finding that rats with pancreatic fistula absorbed 111 mumol of phosphatidylcholine and that controls absorbed 119 mumol of a 135-mumol load. Activity was found to be three times greater in the distal than in the proximal intestine; in cryptal cells it was 10 times greater than in villus tip cells. 65% of the activity in the gut lumen was tightly bound to particulate matter. We propose that intestinal phospholipase may be important in gut bacterial control, in the digestion of vegetable matter (phosphatidylglycerol is a major phospholipid in both plants and bacteria), and in the digestion of phospholipids in the gut lumen.

Effect of acetylglyceryl ether phosphocholine (platelet-activating factor) and thrombin on acylation of exogenous lysophosphoglycerides in rabbit platelets.

Exogenous lysophosphoglycerides such as 1-O-acyl-2-(lyso)-sn-glycero-3-phosphocholine (lysoPC), 1-O-acyl-2-(lyso)-sn-glycero-3-phosphoethanolamine (lysoPE) and 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine (lysoGEPC) radiolabelled on the 1-O-aryl moiety were found to be acylated to the corresponding phosphoglycerides by rabbit platelets at different initial rates and to different extents. LysoPC and lysoGEPC were found to be the best precursors for specifically labelling the corresponding phosphoglyceride pool. Interestingly, synthetic 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and thrombin were able to increase the initial rate of acylation of all these lysophosphoglycerides up to 4-6-fold in a concentration-dependent manner. The finding that stimulation of the acylation rate of the various lysophosphoglycerides was relatively independent of the stimuli used supports the hypothesis that this activation could be a new phenomenon associated with the reversible shape change observed in platelets after stimulation with AGEPC or thrombin.

Importance of human gastric lipase for intestinal lipolysis: an in vitro study.

Using soybean triacylglycerols emulsified with egg lecithin we have studied, in vitro, the influence of substrate prehydrolysis by human gastric lipase upon subsequent degradation by the pancreatic lipase-co-lipase system. Fatty acids liberated by pure human gastric lipase or juice trigger immediate activity of human pancreatic lipase. Gastric lipolysis appears to be of prime importance for dietary lipid digestion in human.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

The influence of bile salts and bile lipoprotein complex on pancreatic lipase hydrolysis of monomolecular films.

We report a new technique which allows us to follow the lipolysis of monomolecular films in the presence of bile salts by using a 'zero-order' trough (Verger, R. and de Haas, G.H. (1973) Chem. Phys. Lipids 10, 127). The effects of bile salts, the bile lipoprotein complex and colipase on pancreatic lipase hydrolysis of rac-1,2-didodecanoylglycerol films were studied at different surface pressures. Taking into account previous studies, lipase activity was interpreted as a function of its degree of binding to the bile lipoprotein complex.

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.

Prethrombotic disorders in children with arterial ischemic stroke and sinovenous thrombosis.

BACKGROUND: Arterial ischemic stroke (AIS) and sinovenous thrombosis (SVT) are relatively rare events in children. The contribution of prethrombotic disorders to the etiology of these entities has not been completely elucidated. OBJECTIVES: To determine the frequency of inherited and acquired prethrombotic disorders in a pediatric population with AIS and SVT and to report clinical and radiological features. METHODS: From May 1992 to April 1997, 30 consecutive children with AIS and 10 children with SVT were assisted at a single institution. Hemostatic evaluation was performed for all the children. Evaluation included the following assays: protein C, protein S, antithrombin, plasminogen, activated protein C resistance, factor V Leiden mutation, and the detection of antiphospholipid antibodies. Data concerning baseline demographics, risk factors, presenting features, family history of thrombosis, and radiological findings were also recorded. RESULTS: One or more prethrombotic disorders were present in 9 children (30%) with AIS (inherited protein S deficiency, 2 patients; inherited protein C deficiency, 1 patient; acquired antithrombin deficiency, 2 patients; antiphospholipid antibodies, 3 patients; and antiphospholipid antibodies and plaminogen deficiency, 1 patient) and in 5 children (50%) with SVT (inherited protein S deficiency, 1 patient; acquired antithrombin deficiency, 3 patients; and antiphospholipid antibodies, 1 patient). CONCLUSIONS: Most children studied presented both a variety of risk factors for thrombosis and concomitant prethrombotic disorders. Therefore, a complete hemostatic evaluation for all children with AIS and SVT should be performed, despite the presence of obvious clinical risk factors or lack of family history of thrombosis.

Acenocoumarol therapy in pediatric patients.

To determine guidelines for administering and monitoring acenocoumarol therapy in children, 93 patients (median 5.1 years, range: 0.2-18 years) were prospectively evaluated over a 33-month period. The loading doses used were: <1 year, 0.20 mg x kg-1; >1-5 years, 0.09 mg x kg-1; 6-10 years, 0.07 mg x kg-1; 11-18 years, 0.06 mg x kg-1. In this study, the loading dose and the dose to achieve and maintain target therapeutic range (TTR) for acenocoumarol are age-dependent, with infants having the highest and teenagers having the lowest requirements. The use of a different loading dose according to age has allowed most of the children (80%) in all the age groups to achieve TTR in less than 1 week. No patients had serious bleeding or thrombotic complications. We conclude that there is an age-dependent response to acenocoumarol in pediatric patients. The implementation of an age-adjusted loading dose regimen reduces the length of hospitalization required to achieve effective anticoagulant therapy.

Glycolipids as potential binding sites for HIV: topology in the sperm plasma membrane in relation to the regulation of membrane fusion.

Although human sperm cells can bind human immunodeficiency virus (HIV-1), they lack CD4, galactoceramides (GalCer) and sulfogalactoceramides (SGalCer) as gp120 receptors. However, sperm specific glycolipids (sulfogalactosylalkylacylglycerol (SGalAAG) and galactosylalkylacylglycerol (GalAAG)) are structurally closely related to SGalCer and GalCer as predicted by computer simulated molecular modelling. SGalAAG and GalAAG are exclusively localized in the outer leaflet of the human sperm plasma membrane, and therefore we tested whether they could serve as alternative receptors for the gp120. Purified SGalAAG and GalAAG had similar affinities to recombinant gp120 as the hydroxy fatty acid (HFA) SGalCer and HFA-GalCer respectively. However, nonhydroxy fatty acid forms of (S)GalCer, galactosyldiacylglycerol and the deacylated (sulfo)galactosyllipids did not recognize recombinant gp120. Data obtained by surface pressure experiments revealed that the lipid monolayers that contained HFA-GalCer or GalAAG resulted in a similar significant penetration of recombinant gp120 in the monolayer. The penetration was a factor of two lower in monolayers with HFA-SGalCer or SGalAAG. The binding of recombinant gp120 to human sperm cells colocalized with GalAAG and could be blocked with monoclonal antibodies against galactolipids. The possible relevance of gp120 binding to glycolipids for HIV entry in sperm cells is discussed.

Behavior of diluted activated partial thromboplastin time in pregnant women with a lupus anticoagulant.

The present study was developed to verify whether a reduction in phospholipid concentration could increase the activated partial thromboplastin time (APTT) sensitivity to detect lupus anticoagulant (LA) during pregnancy. The authors studied 38 pregnant women (10 normal subjects and 28 patients with associated clinical complications) and 40 nonpregnant control subjects. Tests to detect LA, including APTT, platelet neutralization procedure (standard APTT), the kaolin clotting time, the diluted Russell viper venom test neutralized by lysed platelets, and factor assays, were performed. Positive results were found in 5 of 28 pregnant women with associated clinical complications. The APTT, using three different phospholipid concentrations (standard and more diluted cephalin), was performed on plasma samples and on its 1:1 mixture with normal plasma. The behavior of standard and diluted APTT was similar in negative LA pregnant women and nonpregnant control subjects. The mean values showed nonsignificant differences. Four of five pregnant women with positive LA findings had a prolonged APTT, which was not corrected by the addition of normal plasma using standard conditions. When diluted phospholipids were used, only one of them had a prolonged APTT that was corrected by the addition of normal plasma. Therefore, the highest sensitivity (80%) and specificity (100%) of the APTT to detect LA in pregnant women were obtained using the standard conditions.

Immunochemical determination of porcine pancreatic colipase: differentiation between procolipase and its trypsin-activated form.

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative determination of porcine pancreatic colipase. Calibration curves were established by coating polystyrene immunoplates with pure procolipase or its trypsin-activated derivative. Bound antigen was detected with antiporcine procolipase polyclonal antibodies. Under optimizing conditions, the minimal detectable amount of porcine colipase was 0.1 ng, which is about 1,000 times less than the minimal amount that can be assayed titrimetrically. The useful range of the immunoassay was between 0.1 to 1 ng (2-20 micrograms/L). Under standard assay conditions, no distinction can be made between the precursor and activated forms of the cofactor. Results of immunochemical determinations of colipase in porcine pancreatic juice and tissue extract were in good agreement with those obtained with the potentiometric method. The specific determination of activated colipase in pancreatic juice was performed by coating the immunoplates with antigen in solution in PBS with 0.5 g/L of Tween 20. The detergent selectively impaired the binding of procolipase to the plate. Determination of colipase in human pancreatic juice carried out under the same experimental conditions showed that the minimal amount of human cofactor detectable with ELISA was 1 ng due to partial immunological crossreactivity of the human and porcine proteins. Immunoassay performed with antiporcine procolipase monoclonal antibodies (Mab) showed lower sensitivity than that performed with polyclonal antibodies. However, Mab 72.11, a monoclonal antibody that reacted only with porcine procolipase, allowed specific detection and differential determination of the precursor form of porcine colipase in pancreatic juice. ELISA performed with pure human colipase indicated that no antiporcine procolipase monoclonal antibodies cross-reacted with the human cofactor.

A possible orientation change of cardiotoxin molecule during its interaction with phospholipid monolayer.

The monomolecular film technique was used in order to study the specific interactions of 4 cardiotoxins from Elapidae snake venom Naja mossambica mosambica with different phospholipids. The interaction, at pH 7.5, of cardiotoxin (10(-7) M) with both neutral and negatively charged phospholipids occurs up to a very high critical surface pressure (pi = 45 dynes/cm with the latest). The apparent molecular area of cardiotoxin molecule, during its insertion into a negatively charged phospholipid film, presents only two characteristic values: 1400 A2 for pi less than 20 dynes/cm and 420 A2 for pi greater than 30 dynes/cm, the transition occurring in a very narrow range of surface pressure (25 +/- 5 dynes/cm). Thus, according to the surface pressure, the cardiotoxin may present two different orientations: "flat" or "edgewise" and the transition between both could account for lytic activity of cardiotoxin.

Interactions of lipases with lipid monolayers. Facts and questions.

Among the proteins, lipolytic enzymes provide a valuable model for studying protein-lipid interactions. Lipases having a catalytic action which is strictly dependent upon the presence of a lipid interface were used in the present study in order to gain better insight into protein-lipid interactions. Most of the data presented here were obtained using the monolayer technique, by recording (either independently or simultaneously) the lipolytic activity, the amount of protein adsorbed to the lipid monolayer, and the surface pressure variations following protein adsorption. Several non-enzymatic proteins were used as controls in order to determine how lipase behaviour differs from that of other proteins. At all initial surface pressures tested, with zwitterionic monolayers, a good correlation was observed between the amount of lipase bound to the monolayer and the surface pressure increase, in agreement with previous studies. Conversely, with neutral lipid monolayers the amount of lipase bound to the monolayer was not found to be surface pressure dependent. This latter behaviour observed with lipases on neutral films is not specific to lipases, since it was also observed with bovine serum albumin and beta-lactoglobulin A. Lipase activity in the presence of various proteins was investigated with monomolecular films of glycerol didecanoate, either at constant surface area or at constant surface pressure. Depending upon the nature of the lipase and the protein, inhibition of lipase activity was either observed or not. Inhibition was correlated with a decrease in lipase surface concentration. The ability of the various proteins to inhibit lipolysis is: (i) a function of their excess versus lipase in the bulk phase, and: (ii) correlated with their penetration capacity (i.e., the initial rate of surface pressure increase of a glycerol didecanoate monolayer having an initial surface pressure of 20 dyn/cm, after the injection-of the protein). Since lipase inhibition was observed with low surface densities of inhibitory proteins, a long-range effect is probably involved in the mechanism of interfacial lipase inhibition. The nature of the ionic charge added to the monolayer by the protein is not critical for determining lipase adsorption or desorption. It is hypothesized that the lack of lipase adsorption to, or desorption from, the lipid monolayer results from a change in the organization of the hydrocarbon moiety of the lipid.

Observations on the critical micellar concentration of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and a series of its homologs and analogs.

In a study designed to explore the physical chemical characteristics of platelet activating factor (PAF), or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the critical micellar concentration of this compound, as well as the propionyl, butyryl and hexanoyl homologs was determined. In addition, an analogous series of compounds, in which the 1-O-alkyl was replaced by a 1-O-hexadecanoyl or a 1-O-octadecanoyl group, were examined for their critical micellar concentration. A variety of analytical techniques including NMR, gas liquid chromatography, infra-red spectrometry, thin layer chromatography, phosphorus, choline, glyceryl ether and fatty acid analyses were used to confirm the high purity of the individual derivatives. A dye binding assay and a surface tension technique were compared as to their suitability for determination of the critical micellar concentration of these compounds. Whereas the dye binding method proved highly variable, the surface tension procedure proved to be a facile, reproducible technique and was the assay of choice. The critical micellar concentration of the 1-O-alkyl and the 1-O-acyl derivatives showed comparable values for each short chain substituent at carbon 2, with values, in microM, ranging from 1.3 +/- 0.03 for 1-O-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine and 1.1 +/- 0.10 for 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine to 0.22 +/- 0.01 for 1-O-hexadecanoyl-2-hexanoyl-sn-glycero-3-phosphocholine and 0.18 +/- 0.03 for 1-O-hexadecyl-2-hexanoyl-sn-glycero-3-phosphocholine. The data show that at the molar concentration usually employed in biological studies with these compounds, i.e., 1 X 10(-7) to 10(-11) M, one can assume that they will be present as monomolecular species. Thus, it seems unlikely that the widely diverse biological activities of these compounds can be explained by this physical parameter.

Lipase kinetics at the triacylglycerol-water interface using surface tension measurements.

Two methods, the so-called "oil drop" and "Teflon plunger" methods, were designed to monitor lipase hydrolysis of natural long-chain triacylglycerols through the variation with time of the oil-water interfacial tension. The first part of this work is devoted to the development of these two techniques using pure, well-characterized porcine pancreatic lipase. They gave linear responses with enzyme concentrations ranging from 1 x 10(-3) to 30 units x ml-1. We then applied them to a study of the optimal pH conditions for human gastric lipase which were found to range around 5, as previously observed. In the presence of variable concentrations of sodium taurodeoxycholate, these two methods also showed that human gastric lipase is active in the 8-13 dyn cm-1 range of interfacial tension. It is concluded that these two methods, based upon variations with time of the oil-water interfacial tension, constitute reliable, sensitive and convenient means of investigating lipase kinetics.

Altered erythrocyte n-3 fatty acids in Mediterranean patients with coronary artery disease.

A low frequency of ischaemic heart diseases in Eskimos has been related to polyunsaturated fatty acids. We therefore studied fatty acid patterns associated with coronary artery disease (CAD) for a possible relationship between fatty acid profile and CAD diagnosis in Mediterranean patients. The gas chromatography method was used to analyze the membranes of patients' erythrocytes. The patients without coronary stenosis were used as controls. Patients with CAD showed increased percentages of saturated fatty acids (35.8 vs. 34.2%, P<0.001) and monounsaturated fatty acids (14.6 vs. 13.6%, P<0.01), as well as reduced percentages of polyunsaturated fatty acids (38.5 vs. 41.3%, P<0.001). The decrease in polyunsaturated fatty acids percentages was due to the series of n-3 fatty acids (9.2 vs. 11.4%, P<0.001), mainly at the expense of docosahexaenoic acid [C22:6 (n-3)] (4.9+/-0.25% vs. 6.4+/-0.23%, P<0.001) and docosapentaenoic acid [C22:5 (n-3)] (3.0+/-0.19% vs. 3.9+/-0.12%, P<0.001). The study shows altered n-3 fatty acids in Mediterranean patients with CAD. Our data suggest that the percentage of docosahexaenoic and docosapentaenoic acids in erythrocytes could be used as indicators of an independent risk factor for coronary artery disease.

Lipolysis and heterogeneous catalysis. A new concept for expressing the substrate concentration.

A new concept is proposed for quantifying the substrate concentration during heterogeneous catalysis of the kind which occurs during lipolysis. The number of molecules of protein (enzyme) adsorbable to the lipid substrate interface per unit of volume was evaluated and defined as a volumetric concentration of protein (enzyme) binding site (PEBS). Using porcine pancreatic lipase (EC 3.1.1.3) as a model enzyme, the maximal PEBS concentration was measured under various assay conditions by determining the saturation of the lipid substrate with the enzyme. Abacuses correlating the lipid substrate concentration (M) with the PEBS concentration (M) under each experimental conditions were used to express the kinetic data in terms of a volumetric concentration of PEBS. Comparisons could thus be made between data obtained with various enzymes and lipid interfaces because they were expressed with the same unit. In the case of pancreatic lipase, using triolein and tributyrylglycerol as substrates, Km values of 2.7 and 7.5 nM PEBS were obtained, respectively, and KD values ranging around 9 nM PEBS were also obtained from Scatchard plots. In addition, the average superficial density of PEBS was found to be 10 x 10(11) molecules.cm-2, which is a value commonly obtained with structural proteins and enzymes adsorbed to an acylglyceride-water interface, this finding supports the idea that the PEBS concept represents the room in which the protein molecule adsorbs at the lipidic interface.

A graph-theoretic method for decomposing two-dimensional polygonal shapes into meaningful parts.

This note gives a new graph-theoretic shape decomposition procedure, extending the idea in the paper ``Decomposition of Two-Dimensional Shapes by Graph-Theoretic Clustering'' by Shapiro and Haralick [1]. A binary matrix is used in order to express the LI relation. A solution for the problems connected with artificial separation of two CS cliques and the presence of a regular concavity is proposed. Our algorithm produces nonoverlapping shape parts in much reduced execution time.