Biomedical subjects

C Garbay

Publications and source records attributed to C Garbay.

At least 37 records · Page 2Linked to original sources

PubMed1998

Differential interactions of the growth factor receptor-bound protein 2 N-SH3 domain with son of sevenless and dynamin. Potential role in the Ras-dependent signaling pathway.

In this paper, we show that the 36-45 surface-exposed sequence WYKAELNGKD of growth factor receptor-bound protein 2 (Grb2) N-SH3 domain inhibits the interaction between Grb2 and a 97-kDa protein identified as dynamin. Moreover, the peptide GPPPQVPSRPNR from dynamin also blocks the binding of dynamin to the proline-rich recognition platform of Grb2. Mutations in the 36-45 motif show that Glu-40 is critical for dynamin recognition. These observations were confirmed by immunoprecipitation experiments, carried out using ER 22 cells. It was also observed that the proline-rich peptide from dynamin was unable to dissociate the Grb2.Sos complex, whereas the proline-rich peptide from Son of sevenless (Sos) inhibited Grb2. dynamin interaction. A time-dependent stimulation of epidermal growth factor receptor overexpressing clone 22 (ER 22) cells by epidermal growth factor resulted in an immediate increase of the Grb2.Sos complex and a concomitant decrease in Grb2.dynamin. This suggests that the recruitment of Grb2.Sos to the membrane, triggered by epidermal growth factor stimulation, activates the Ras-dependent signaling and simultaneously enhances free dynamin levels, leading to both receptor internalization and endocytotic processes.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Interleukin-2 stimulation induces tyrosine phosphorylation of p120-Cbl and CrkL and formation of multimolecular signaling complexes in T lymphocytes and natural killer cells.

Interleukin (IL)-2, a major growth and differentiation factor for T lymphocytes, was found to induce tyrosine phosphorylation of the proto-oncogene products p120-Cbl and CrkL in IL-2-dependent cell lines. We established that, in unstimulated lymphocytes, the Src homology 2 (SH2) and SH3 domain-containing protein Grb2 and the p85 subunit of phosphatidylinositol 3-kinase, associate constitutively with Cbl via their SH3 domains. Furthermore, IL-2 stimulation increased the level of interaction of phosphorylated Cbl with the p85 SH2 domains, and we provide evidence that the preformed Cbl-Grb2 complex recruits the phosphorylated p52 Shc adaptor protein. In addition, we demonstrate that the SH2-SH3-SH3 adaptor protein CrkL is tyrosine-phosphorylated in an IL-2-dependent manner and, via its SH2 domain, associates with a large proportion of phosphorylated Cbl. We also show that p85 is preassociated with the CrkL SH3 domain. Furthermore, the association of CrkL and p85 is increased after IL-2 treatment by a mechanism involving intermediary tyrosine-phosphorylated proteins that remain to be identified. Our results show that CrkL associates independently with Cbl or p85 and suggest that it also participates in larger complexes containing Cbl and p85. Although the precise roles of Cbl and CrkL remain to be elucidated, their tyrosine phosphorylation, in addition to the multiple protein interactions described here, strongly suggest that Cbl and CrkL may play pivotal roles in the early steps of IL-2 signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Opposite effects of CCK(B) agonists in grooming behaviour in rats: further evidence for two CCK(B) subsites.

1. The hypothesis of the existence of two CCK(B) receptor subsites, CCK(B1) and CCK(B2) corresponding probably to different coupling states of CCK(B) receptors, was studied by measuring grooming behaviour in rats. 2. The B1 receptor agonist, BC197 (300 microg kg(-1), i.p.) produced a 45-50% decrease in grooming activity, which was prevented by both the CCK(B) receptor antagonists CI-988 (20 microg kg(-1) i.p.) and L-365,260 (200 microg kg(-1), i.p.). 3. In contrast, 3, 10 and 30 microg kg(-1), i.p., of the potent B2 receptor agonist, BC264, enhanced grooming (150-190%). This effect was prevented by previous injection of 75 microg kg(-1) of L-365,260 while higher doses (200 microg kg(-1), i.p.) produced only a partial antagonism. Moreover, CI-988 (20 microg kg(-1), i.p.), showed an opposite effect in potentiating the responses induced by BC264. However, 200 microg kg(-1) of CI-988 tended to suppress the increase of grooming induced by BC264. 4. The effects of BC264 were prevented by the D1 receptor (SCH 23390) and D2 receptor (sulpiride) antagonists, while those of BC197 were only antagonized by sulpiride, emphasizing the existence of a link between peptidergic (CCK) and dopaminergic systems. 5. This study brings additional evidence for the existence of the two CCK(B) receptor subsites and suggests that particular attention should be focused on the selectivity of CCK(B) receptor agonists, notably to explain the fact that some compounds such as Boc-CCK4 induce anxiogenic-like effects while others, including BC264, are devoid of these effects.

Animals↗

PubMed1997

Synthesis and biological properties of new constrained CCK-B antagonists: discrimination of two affinity states of the CCK-B receptor on transfected CHO cells.

To improve our knowledge of the bioactive conformation of CCK-B antagonists, we have developed a new series of constrained dipeptoids whose synthesis and biochemical properties are reported here. These compounds, of general structure N alpha-[(2-adamantyloxy)carbonyl]-alpha-methyltryptophanyl-(4 -X)-proline, were designed by introducing a cyclization in the structure of the previously described CCK-B/peptoid antagonist RB 210, N-[N-[(2-adamantyloxy)carbonyl]-DL-alpha-methyltryptophanyl] -N-(2-phenylethyl)glycine (Blommaert et al. J. Med. Chem. 1993, 36, 2868-2877), by means of a five-membered ring. Structure-affinity relationship studies showed that an R configuration of Trp-C alpha and a cis configuration of the pyrrolidine substituents were favorable for receptor recognition. The most potent compounds of this new series had similar affinities for the CCK-B receptor as RB 210 and proved to be far more efficient in inhibiting inositol phosphate production in CHO cells stably transfected with rat brain CCK-B receptor, with IC50 values approaching those of the commonly used antagonists L-365,260 and PD-134,308. Moreover, binding studies performed using transfected CHO cells showed that two affinity states of the CCK-B receptor can be discriminated by some of these compounds which also have different biological profiles and are therefore highly interesting tools for the biochemical and pharmacological characterization of CCK-B receptor heterogeneity.

Adamantane↗

PubMed1997

Structure-based design of new constrained cyclic agonists of the cholecystokinin CCK-B receptor.

New constrained cyclic pseudopeptide cholecystokinin-B (CCK-B) agonists have been designed on the basis of conformational characteristics of the potent and selective CCK-B agonist Boc-Trp-(NMe)Nle-Asp-Phe-NH2 (Ki = 0.8 nM, selectivity ratio CCK-A/CCK-B > 6000) (Goudreau et al. Biopolymers, 1994, 34, 155-169). These compounds are among the first successful examples of macrocyclic constrained CCK4 analogs endowed with agonist properties and as such may be of value for the development of nonpeptide CCK-B agonists. The affinities and selectivities of these compounds for CCK-B and CCK-A receptors have been determined in vitro by measuring the displacement of [3H]pCCK8 binding to guinea pig cortex and pancreas membranes, respectively. The most potent compound, 8b, N-(cycloamido)-alpha-Me(R)Trp-[(2S)-2-amino-9- ((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH2, has a Ki value of 15 +/- 1 nM for guinea pig cortex membranes with a good CCK-B selectivity ratio (CCK-A/CCK-B = 147). Furthermore, 8b behaved as a potent and full agonist in a functional assay which measures the stimulation of inositol phosphate accumulation in CHO cells transfected with the rat CCK-B receptor (EC50 = 7 nM). The in vivo affinity of 8b for mouse brain CCK-B receptors was determined following intracerebroventricular injection (ID50 approximately 29 nmol/kg). 8b was also shown to cross the blood-brain barrier (0.16%), after intravenous administration in mice. 8b also increased gastric acid secretion measured in anesthetized rats after intravenous injection. Therefore, 8b appears to be an interesting pharmacological tool and is currently under investigation as a lead for further development of nonpeptide CCK-B agonists.

Animals↗

PubMed1996

Novel CCK-B receptor agonists: diketopiperazine analogues derived for CCK4 bioactive conformation.

Recently, we proposed a CCK-B agonist bioactive conformation characterized by an 'S' shape of the peptidic backbone which was derived from structure-activity relationships and conformational analysis of CCK4 (Trp-Met-Asp-Phe-NH2) analogues. Using this template, we report here the synthesis of cyclic CCK4 analogues which contain, in place of the Trp-Met dipeptide, a diketopiperazine moiety resulting from a cyclization between Nle and N-substituted (D)Trp residues and coupled with a small linker to Asp-Phe-NH2. Some of these compounds displayed good affinities and selectivities for the CCK-B receptor. The results are discussed in terms of size, hydrophobicity and spatial orientation of the side-chains on the diketopiperazine ring. The most potent ligand exhibited potent and full CCK-B receptor agonist properties in promoting the hydrolysis of inositol phosphates (EC50 = 8 nM) in CHO cells, stably transfected with the rat brain CCK-B receptor. This compound was also shown to be a potent selective CCK-B/gastrin receptor agonist since, it increased gastric acid secretion measured in anesthetized rats on i.v. administration. These compounds provide a rigid template for the design of non-peptide CCK-B agonists, by modification of the remaining peptide moiety.

Animals↗

PubMed1996

Role of N- and C-terminal substituents on the CCK-B agonist-antagonist pharmacological profile of Boc-Trp-Phg-Asp-Nal-NH2 derivatives.

Among the CCK derivatives, the tetrapeptide Boc-Trp-Phg-Asp-Nal-NH2 (1) behaves as a short potent CCK-B agonist which led to the development of an efficient peptidase-resistant CCK-B antagonist by bismethylation of its terminal CONH2 group. Further modifications of the N- and C-terminal moieties of 1 have been performed and are described in this paper, together with the pharmacological profile of the novel synthetized compounds. Introduction of more bulky substituents than NalNH2 on the C-terminal part decreased the CCK-B receptor binding affinity. In the series of N-protected tetrapeptides X30-Phg31-Asp32-Nal33-N(CH3)2, the Boc-substituent was shown to be optimal among the N-protecting groups Boc, 2Adoc, propionyl or acetyl when X = Trp. On the other hand, when X = alpha MeTrp, its optimal N-protecting group was 2Adoc and its configuration was preferentially D. In the newly synthesized compounds, 13: 2Adoc-D-alpha MeTrp-Phg-Asp-NalN(CH3)2 and 16: 2Adoc-D-alpha MeTrp-Phg-Asp-NalNH2 had the best CCK-B receptor affinities (KI = 3.5 and 3.4 nM, respectively) and were selected for further biological evaluation. Interestingly, when tested for their capacity to influence inositol phosphate formation, induced by CCK8 in CHO cells transfected with the rat CCK-B receptor, compound 13 behaved as a full CCK-B antagonist with an IC50 value of 18 +/- 1 nM, being as potent as the antagonist L-365,260 and PD-134,308 (IC50 values respectively, 39 +/- 17 and 30 +/- 2 nM), whereas compound 16 was found to behave as a partial CCK-B agonist. Indeed 16 behaved as an antagonist on the firing rate of rat CA1 hippocampal neurons and acted as an agonist in the pentagastrin stimulated gastric acid secretion (EC50 = 12 nmol/kg) in anesthetized rats. Compound 13 in contrast, was found to inhibit the pentagastrin action at a dose (ID50 = 0.56 mumol/kg) similar to the potent antagonist PD-134,308 (ID50 = 0.4 mumol/kg). The antagonist/agonist properties of compounds 13 and 16 show that both N- and C-terminal substituents modulate the pharmacological properties in the Boc-CCK4 derivatives presented here.

Amino Acid Sequence↗

PubMed1996

Synthesis and biological evaluation of a new series of phenylhydroquinone derivatives as inhibitors of EGF-R-associated PTK activity.

In order to design new potent inhibitors of the epidermal growth factor receptor (EGF-R) associated protein tyrosine kinase (PTK) activity as antitumor agents, several families of phenylhydroquinone derivatives were synthesized. Some of these compounds were shown to block PTK activity in vitro, but also efficiently to inhibit EGF-stimulated DNA synthesis in ER 22 cells (CCL 39 hamster fibroblasts transfected with EGF-R) with IC50 values in the range 1-10 microM. In some cases, a correlation between the two sets of data was observed, allowing structure-activity relationships to be established. However, inhibitors which had in vitro specificity with regard to other kinases were not specific in the cellular test. Similar effects on DNA synthesis were observed after stimulation by various activating agents, suggesting that our compounds may also act against other cellular targets involved in the EGF-dependent pathways leading to cell proliferation.

Amino Acid Sequence↗

PubMed1996

Synthesis and biological evaluation of a series of hydroxybenzylphenylamine derivatives as inhibitors of EGF receptor-associated tyrosine kinase activity.

In order to obtain non-degradable and more potent protein-tyrosine kinase inhibitors, derived from the 5-(2,5-dihydroxybenzyl)-aminosalicylates already described, we have developed a new series of 5-(2,5-dihydroxybenzyl)phenylamines. The compounds, diversely substituted on the phenyl ring by alcohol, nitrile, ether, ketone, amide and thioamide groups, were tested for their ability to inhibit epidermal growth factor (EGF) receptor-associated tyrosine kinase activity in vitro. They inhibit the phosphorylation of the peptide substrate RR-Src by the EGF receptor purified from ER 22 cells, with IC50 values in the range 0.02-0.45 microns. Several of these compounds inhibit EGF-dependent DNA synthesis in ER 22 cells with IC50 values of around 1 micron and furthermore their inhibition has been found to be specific for various protein kinases.

Aniline Compounds↗

PubMed1995

Inhibition of the EGF-stimulated cellular proliferation of ER 22 cells by hydroxybiphenyl derivatives.

Several series of hydroxybiphenyl compounds substituted by a hydrophobic group (tert-butyl or phenyl) and bearing a free or protected carboxylic moiety were synthesized. The compounds were tested for their ability to inhibit the intrinsic tyrosine protein kinase activity of the EGF-receptor in vitro and the EGF-stimulated DNA-synthesis by ER 22 cells. Although the compounds of each series had poor in vitro inhibitory potencies (IC50 >> 100 microM), most of them inhibited the EGF-dependent cellular proliferation of ER 22 cells at relatively low doses (IC50 = 1.1 microM for compound 14). Structure-activity studies based on the cellular results showed that the most interesting series was the linear terphenyl series B of 2'-hydroxy-1,1':4',1"-terphenyl-4-carboxylates. The availability of the hydroxyl group, either protected or unprotected, the linear arrangement of the hydrophobic moiety, the biphenyl skeleton, and the carboxylic group seem to be essential for the activity of the compounds.

Amino Acid Sequence↗

PubMed1995

Protein kinases in the locus coeruleus and periaqueductal gray matter are involved in the expression of opiate withdrawal.

The aim of this study was to evaluate the role played in the behavioral expression of morphine withdrawal syndrome by protein kinases in the locus coeruleus and the periaqueductal gray matter. Two different families of specific protein kinases have been investigated: serine/threonine and tyrosine kinases. Rats were implanted with cannulas into both the lateral ventricle and the locus coeruleus or the periaqueductal gray matter. Physical dependence was induced by chronic peripheral administration of morphine (from 7 to 30 mg/kg) and withdrawal syndrome was precipitated by injection of naloxone (2 micrograms) into the lateral ventricle. The administration of the serine/threonine kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, H7 (1, 3, 10, and 30 nmol per side) into the locus coeruleus induced a strong attenuation of morphine withdrawal behavior. Signs related to the motor component of abstinence, such as jumping, rearing, and hyperactivity, were the most severely reduced. However, this effect was not dose-dependent, and the response was almost the same with all the doses used. A similar attenuation was observed after the injection of H7 (1, 3, and 10 nmol per side) into the periaqueductal gray matter, but in this case motor signs were less strongly reduced and a larger number of signs were modified, mainly when using the highest dose. The administration of the tyrosine kinase inhibitor 2-hydroxy-5-[N(2,5-dihydroxyphenyl)methyl]amino]-benzoic acid 3-phenylpropyl ester, KB23 (0.3, 1, and 3 nmol per side) into the locus coeruleus or the periaqueductal gray matter had no effect on the withdrawal syndrome behavior, except on teeth chattering. These results suggest that in the locus coeruleus and in the periaqueductal gray matter, serine/threonine kinases are implicated in the behavioral expression of morphine abstinence. In these brain structures, tyrosine kinases appear not to be involved.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Structure-activity relationships in a series of 5-[(2,5-dihydroxybenzyl)amino]salicylate inhibitors of EGF-receptor-associated tyrosine kinase: importance of additional hydrophobic aromatic interactions.

Potent inhibitors of EGF-dependent protein tyrosine kinase (PTK) activity were synthesized in a series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Several of these compounds inhibited EGF-dependent DNA synthesis in ER 22 cells with IC50 < 1 microM. In this series of PTK inhibitors, the role of the salicylate moiety as a potential divalent ion chelator was tested and found to be nonessential in all cases. The length and ramification of the substituting carboxyl group were investigated to improve cellular bioavailability, and this analysis provided compounds with increased inhibitory effect on EGF-induced DNA synthesis. Salicylates esterified with long hydrophobic chains were shown to be noncompetitive inhibitors of ATP, in contrast to the free acid and methyl salicylate. Moreover, all the tested inhibitors were shown to be noncompetitive inhibitors of the peptide substrate. Structure-activity relationships allowed us to suspect a hydrophobic pocket in the tyrosine kinase domain, preferentially interacting with aromatic rings. Finally, the selectivity of the best inhibitors was tested against other kinases, and they were found to be selective for tyrosine kinase. They were also shown to be good inhibitors of EGF-receptor autophosphorylation.

Animals↗

PubMed1994

Solution structure of GAP SH3 domain by 1H NMR and spatial arrangement of essential Ras signaling-involved sequence.

Src homology 3 (SH3) domains are found in numerous cytoplasmic proteins involved in intracellular signal transduction. We used 2-D 1H NMR to determine the structure of the SH3 domain of the guanosine triphosphatase-activating protein (GAP), an essential component of the Ras signaling pathway. The structure of the GAP SH3 domain (275-350) was found to be a compact beta-barrel made of six antiparallel beta-strands arranged in two roughly perpendicular beta-sheets with the acidic residues located at the surface of the protein. The Trp317, Trp319, Thr321 and Leu323 residues belonging to the sequence (317-326), which was shown to be essential for Ras signaling, formed two nearby lipophilic bulges followed by a hydrophilic domain (Arg324-Asp326). These structural data could be used to characterize the still unidentified downstream components of GAP, which are involved in Ras signaling, and to rationally design inhibitors of this pathway.

Amino Acid Sequence↗

PubMed1994

NMR structure of the N-terminal SH3 domain of GRB2 and its complex with a proline-rich peptide from Sos.

GRB2 is a small adaptor protein of 217 amino acids comprising one SH2 domain surrounded by two SH3 domains. GRB2 couples receptor tyrosine kinase activation to Ras signalling by interacting, through its SH3 domains, to the carboxy-terminal proline-rich regions of the guanine nucleotide exchange factor Sos. Here we report the synthesis and solution structure of the amino-terminal SH3 domain of GRB2 and of its more stable Ser 32 mutant. 1H NMR analysis of the complex between the Ser-32-GRB2-N-SH3 domain and the proline-rich peptide VPPPVPPRRR, derived from h-Sos, shows that relative to the SH3 peptide complexes described for PI3K, Fyn and Abl, the proline-rich peptide in this complex binds in the opposite orientation.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Theoretical design, chemical synthesis and footprinting analysis of a novel peptide derivative of the intercalator 7-H pyridocarbazole targeted towards the major groove of DNA.

In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisintercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated beta-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pyrpur) sequences, d(CpG)2 and d(CpA).d(TpG); second, into the (pur-pyr) sequences, d(GpC)2 and d(GpT).d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG).d(CGT) and d(ACA)d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C = O and NH groups of the D-Asn side chain and HN6 and N7 (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N7 of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N7 and O6 of the 3' guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.

Base Sequence↗

PubMed1990

Low concentrations of acridine dimers inhibit micrococcus AP endonuclease through interaction with apurinic sites in DNA.

The effect of dimeric DNA intercalating compounds was assayed on a purified AP endonuclease from Microccoccus luteus using apurinic supercoiled PM2 DNA as a substrate. Binding on apurinic sites was estimated through the competition with the intercalating compound, 9-NH2-ellipticine, which displays great specificity for apurinic sites. An acridine dimer with a spermine linker is at 0.1 microM the best inhibitor of cleavage at the apurinic site induced either by the AP endonuclease or by 9-NH2-ellipticine. Bisintercalating agents are more effective inhibitors of AP endonuclease than monointercalating ones. Most effective inhibitors among dimers have acridine residues.

Acridines↗

PubMed1987

Geometry of the antitumor drug ditercalinium bisintercalated into d(CpGpCpG)2 by 1H NMR.

Rigid 7H-pyrido[4,3-c]carbazole dimers, such as Ditercalinium, are DNA bisintercalators that display high DNA affinity and strong antitumor properties. This activity appears crucially dependent on the geometry of their complexes with DNA. Therefore, structures of the complexes formed by the self-complementary tetranucleotide d(CpGpCpG) with Ditercalinium and with a related monomer were investigated in 0.1 M [2H]acetate buffer (pH 5.5) by using 400-MHz 1H NMR. In both cases, d(CpGpCpG) retained a right-handed duplex structure as shown by exchangeable-proton analysis and intramolecular nuclear Overhauser effect measurements. According to the large upfield shifts measured on the base protons (including the imino proton) and on the aromatic protons of the pyridocarbazole rings, the monomer appears to monointercalate and the dimer to bisintercalate into the tetranucleotide duplex. Ditercalinium dissociates from its complex about 100-1000 times slower than does the monomer. The negative intermolecular nuclear Overhauser effects observed on protons corresponding to the convex edge of the pyridocarbazole rings when the sugar protons are saturated suggest that both ligands intercalate with their chain oriented to the wide groove side of the helix, a situation mimicking that encountered with repressors. Antitumor activity of 7H-pyridocarbazole derivatives is discussed in terms of geometry of the intercalated complexes.

Antineoplastic Agents↗

PubMed1986

An iterative region-growing process for cell image segmentation based on local color similarity and global shape criteria.

An image segmentation process was derived from an image model that assumed that cell images represent objects having characteristic relationships, limited shape properties and definite local color features. These assumptions allowed the design of a region-growing process in which the color features were used to iteratively aggregate image points in alternation with a test of the convexity of the aggregate obtained. The combination of both local and global criteria allowed the self-adaptation of the algorithm to segmentation difficulties and led to a self-assessment of the adequacy of the final segmentation result. The quality of the segmentation was evaluated by visual control of the match between cell images and the corresponding segmentation masks proposed by the algorithm. A comparison between this region-growing process and the conventional gray-level thresholding is illustrated. A field test involving 700 bone marrow cells, randomly selected from May-Grünwald-Giemsa-stained smears, allowed the evaluation of the efficiency, effectiveness and confidence of the algorithm: 96% of the cells were evaluated as correctly segmented by the algorithm's self-assessment of adequacy, with a 98% confidence. The principles of the other major segmentation algorithms are also reviewed.

Biometry↗