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PubMed2000

Microanalysis of melanins in mammalian hair by alkaline hydrogen peroxide degradation: identification of a new structural marker of pheomelanins.

A highly sensitive, easy-to-perform method for melanin analysis in pigmented tissues based on alkaline hydrogen peroxide degradation has been developed and accomplishes simultaneous determination of eumelanins and pheomelanins. Pyrrole-2,3,5- tricarboxylic acid, the typical eumelanin marker, was obtained in higher yields than in previous procedures. A benzothiazole acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, characterized in our previous studies as a specific marker of pheomelanins, and the newly identified 1,3-thiazole-2,4, 5-tricarboxylic acid were also used for pigment analysis. Optimal yields of the pigment markers were obtained at 24 h reaction time. Pyrrole-2,3,5-tricarboxylic acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, and 1, 3-thiazole-2,4,5- tricarboxylic acid were quantified in a single chromatographic analysis without fractionation or work up of the degradation mixture. The linearity (linearity coefficient from 0.997 to 0.999) was excellent and the inter-assay (percentage coefficient of variation values in the range 0.2-2, n = 6) and intra-assay (percentage coefficient of variation values </= 0.4) reproducibility of the method was very satisfactory. A variety of mammalian hairs including coat color mutant mice and human hairs were used to evaluate the method. All the phenotypically pheomelanic hairs gave 1, 3-thiazole-2,4,5-tricarboxylic acid in variable yields, whereas 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole was obtained only from some of the red hair samples examined. Notably, a marked tendency to actinic damage, as evidenced by determination of the minimal erythema dose, was exhibited by those subjects whose hair afforded the highest 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole yields. This suggests that 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole represents a new biogenetic marker for predicting individuals at high risk for skin cancer and melanoma.

Animals↗
PubMed2000

Newer substituted beta-aminonaphthalenes as potent anti-inflammatory agents.

beta-[2-(3'-Chloro-2'-oxo-4'-substituted aryl-1'-azetidinyl)-thiazol-4- yl]aminonaphthalenes 13-17 and beta-[2-(1',3'-disubstitutedphenyl-formazan-4'-yl)-thiazol-4-yl] aminonaphthalenes 8-12 were synthesized from beta-(2-arylideneamino-thiazol-4-yl)aminonaphthalenes 3-7 by diazotisation and by cycloaddition with monochloroacetyl chloride, respectively, on the azomethine group of the compounds 3-7. The newly synthesized compounds showed potent anti-inflammatory and analgesic activities and were less ulcerogenic than phenylbutazone.

2-Naphthylamine↗
PubMed1995

Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.

We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.

Base Sequence↗
PubMed1995

Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor.

Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

Amino Acid Sequence↗
PubMed1976

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.

Adenosine↗
PubMed1997

Expression of thiamin biosynthetic genes (thiCOGE) and production of symbiotic terminal oxidase cbb3 in Rhizobium etli.

In this paper we report the cloning and sequence analysis of four genes, located on plasmid pb, which are involved in the synthesis of thiamin in Rhizobium etli (thiC, thiO, thiG, and thiE). Two precursors, 4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethylpyrimidine pyrophosphate, are coupled to form thiamin monophosphate, which is then phosphorylated to make thiamin pyrophosphate. The first open reading frame (ORF) product, of 610 residues, has significant homology (69% identity) with the product of thiC from Escherichia coli, which is involved in the synthesis of hydroxymethylpyrimidine. The second ORF product, of 327 residues, is the product of a novel gene denoted thiO. A protein motif involved in flavin adenine dinucleotide binding was found in the amino-terminal part of ThiO; also, residues involved in the catalytic site of D-amino acid oxidases are conserved in ThiO, suggesting that it catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. The third ORF product, of 323 residues, has significant homology (38% identity) with ThiG from E. coli, which is involved in the synthesis of the thiazole. The fourth ORF product, of 204 residues, has significant homology (47% identity) with the product of thiE from E. coli, which is involved in the condensation of hydroxymethylpyrimidine and thiazole. Strain CFN037 is an R. etli mutant induced by a single Tn5mob insertion in the promoter region of the thiCOGE gene cluster. The Tn5mob insertion in CFN037 occurred within a 39-bp region which is highly conserved in all of the thiC promoters analyzed and promotes constitutive expression of thiC. Primer extension analysis showed that thiC transcription in strain CFN037 originates within the Tn5 element. Analysis of c-type protein content and expression of the fixNOQP operon, which codes for the symbiotic terminal oxidase cbb3, revealed that CFN037 produces the cbb3 terminal oxidase. These data show a direct relationship between expression of thiC and production of the cbb3 terminal oxidase. This is consistent with the proposition that a purine-related metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.

Amino Acid Sequence↗
PubMed1997

Characterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis.

The characterization of a three-gene operon (the thiC operon) at 331 min, which is involved in thiamine biosynthesis in Bacillus subtilis, is described. The first gene in the operon is homologous to transcription activators in the lysR family. The second and third genes (thiK and thiC) have been subcloned and overexpressed in Escherichia coli. ThiK (30 kDa) catalyzes the phosphorylation of 4-methyl-5-(beta-hydroxyethyl)thiazole. ThiC (27 kDa) catalyzes the substitution of the pyrophosphate of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate to yield thiamine phosphate. Transcription of the thiC operon is not regulated by thiamine or 2-methyl-4-amino-5-hydroxymethylpyrimidine and is only slightly repressed by 4-methyl-5-(beta-hydroxyethyl)thiazole.

Amino Acid Sequence↗
PubMed1987

Pharmacological properties of the novel anti-platelet aggregating agent 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid.

Various pharmacological properties of a new antiplatelet aggregating agent, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid (E-5510), were examined in order to elucidate its mode of action, Firstly, the inhibitory effect on in vitro aggregation of platelets from humans and various experimental animals was studied. E-5510 inhibited human platelet aggregation induced by collagen, arachidonic acid, adenosine diphosphate (ADP), platelet activating factor (PAF) and epinephrine. Thrombin-induced platelet aggregation, which was not inhibited by acetylsalicylic acid (ASA) or the thiazole drug, 4,5-bis(4-methoxyphenyl)-2-(trifluoromethyl) thiazole, was inhibited by E-5510. E-5510 inhibited collagen-induced platelet aggregation in platelet-rich plasma (PRP) from guinea pigs, beagle dogs and monkey to the same degree as in human PRP, but its effect was weaker in rat PRP. Human platelet adhesion to a collagen-coated plastic disk and thrombin-induced adenosine triphosphate (ATP) release from human platelets were also inhibited by this compound. Next, the ex vivo anti-platelet effect of E-5510 was examined in guinea pigs and beagle dogs. E-5510 was the most potent among the tested drugs (ticlopidine, ASA, cilostazol and the thiazole drug. The anti-platelet effect of this compound appeared within 1 h and lasted more than 8 h after oral administration. The above results suggest that E-5510 may antagonize platelet activation by inhibiting phospholipase C and/or A2, which results in suppression of both phosphatidylinositol breakdown and arachidonic acid release from phospholipids, as well as by inhibiting cyclooxygenase. E-5510 exerted its anti-platelet action without affecting prostaglandin I2 production in the blood vessels. It is considered that E-5510 has a highly potent anti-platelet aggregating effect and a unique multi-site mode of action. This compound is a promising candidate as an antithrombotic drug for clinical use.

Animals↗
PubMed1993

Biochemical consequences of resistance to tiazofurin in human myelogenous leukemic K562 cells.

Tiazofurin exhibits antitumor activity in murine and human tumor cells. In a recent phase I/II trial in patients with end-stage leukemia, tiazofurin showed good response; however, repeated treatment resulted in clinical resistance to the drug. To elucidate the mechanisms of resistance in human leukemic cells, two variants of human myelogenous leukemia K652 cells resistant to tiazofurin were developed by drug-selection pressure. Compared to a concentration producing 50% cell proliferation reduction that was 9.1 microM in sensitive cells, the resistant variants displayed concentrations producing 50% cell proliferation reductions of 12 and 16 mM. The activity of the target enzyme, IMP dehydrogenase, was not altered in the resistant cells. Studies on tiazofurin metabolism revealed that resistant variants formed < 10% of the active metabolite, thiazole-4-carboxamide adenine dinucleotide. This correlated with the activity of NAD pyrophosphorylase, the enzyme that synthesizes thiazole-4-carboxamide adenine dinucleotide, which was reduced to 10% in the resistant lines. Concurrently, the activity of thiazole-4-carboxamide adenine dinucleotide phosphodiesterase was elevated in the refractory cells. Compared to the sensitive counterpart, the levels of GMP and NAD were lower in the resistant lines. Guanine salvage activity was decreased in the resistant cells. Basal dGTP and dATP concentrations were elevated in the resistant line; nevertheless, tiazofurin incubation decreased dGTP levels in only the sensitive cells. Although there was no difference in the Km of tiazofurin transport or efflux, the Vmax of uptake of the drug was reduced in the resistant lines. Sensitive and resistant cells exhibit similar cytotoxicity to agents which do not share the mechanism of action of tiazofurin, suggesting that refractory cells are still sensitive to other standard antileukemic drugs.

Adenine Nucleotides↗
PubMed1993

Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor.

The gel mobility-shift assay is an important tool for the study of protein-nucleic acid interactions. High detection sensitivity is typically attained by radioisotopic labeling of the target nucleic acid fragments. A novel fluorescence methodology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole yellow homodimers form stable, highly fluorescent complexes with double-stranded DNA that can be detected in gels by a laser-excited, confocal, fluorescence scanning system with a sensitivity higher than that attainable with radioisotopic labeling. We describe here the use of these dyes in a gel-mobility assay to detect complexes of a truncation of the Kluyveromyces lactis heat shock transcription factor, containing the trimerization and DNA-binding domains (HSFDT), with target DNA. At an appropriate molar DNA base pair to dye ratio, the labeling of a DNA fragment with dimeric dye did not affect the binding to HSFDT. The detection of the fluorescent-dye labeled HSFDT-DNA complexes with the laser scanner achieves a spatial resolution far superior to that of conventional autoradiography and permits analysis of multimer protein-DNA complexes that are not resolved by traditional detection methods. We have used this technique to demonstrate that HSF forms multimeric complexes on DNA by addition of trimeric units. The latter conclusion is based on an analysis of the mobilities of the multiple HSFDT-DNA complexes and on a two-color mobility-shift fluorescence assay that uses a mutant of HSFDT engineered for site-specific labeling with fluorescein and target DNA labeled with an "energy transfer" dye, thiazole orange-thiazole blue heterodimer.

Base Sequence↗
PubMed1999

Characterization of ionized heterocyclic carbenes by ion-molecule reactions.

1,2-Hydrogen shift isomers of ionized pyridine, thiazole and imidazole are readily characterized by the study of their associative ion-molecule reactions with dimethyl disulfide in the quadrupole collision cell of a new hybrid sector-quadrupole-sector mass spectrometer. Efficient trapping reactions of CH(3)S(.) radicals are indeed observed and the actual structure of the adduct [M + CH(3)S](+) ions is clearly indicated by their high-energy collisional activation mass spectra. These trapping reactions are not observed for the 'conventional' pyridine, thiazole and imidazole molecular ions, which only react by charge exchange producing m/z 94, [CH(3)SSCH(3)](*+), ions.

Free Radicals↗
PubMed2004

Synthesis and evaluation of some novel quinazolinone derivatives as diuretic agents.

A new series of quinazolin-4(3H)-one derivatives containing either a thiazole or a 1, 3, 4-thiadiazole moiety were prepared in order to study the effect of such a heterocyclic combination on the expected diuretic activity. Synthesis of the target compounds (2, 4, and 6) has been achieved through an interaction of the starting 7-chloro-2-methyl-4H-3, 1-benzoxazin-4-one 1 with different heterocyclic amines. Alkylation of 3-(2-mercapto-1, 3, 4-thiadiazol-5-yl)quinazolin-4(3H)-one derivative 4 with different alkyl halides or chloroacetic acid afforded the corresponding thioethers 5 while interaction of 2-methyl-3-(1, 3, 4-thiadiazol-5-yl or thiazol-5-yl)quinazolin-4(3H)-ones (2 and 6) with various aromatic aldehydes resulted in the formation of the arylvinyl analogs 3 and 7, respectively. On the other hand, 2-morpholinomethyl-3-(2-sulfamoyl or mercapto-1, 3, 4-thiadiazol-5-yl)quinazolin-4(3H)-one derivatives 10 have also been synthesized through an interaction of the sulfonamide or thiol analog 9 with the appropriate amine. Biological evaluation of some of the target compounds as diuretic agents was carried out. The results showed that 2-[2-(4-chlorophenyl)vinyl]-7-chloro-3-(2-sulfamoyl-1, 3, 4-thiadiazol-5-yl)quinazolin-4(3H)-one 7b exhibited significant diuretic activity. The detailed synthesis, spectroscopic and biological data are reported.

Animals↗
PubMed1995

Contribution of mass spectrometry to the structural confirmation of components of the antibiotic GE2270 complex.

The GE2270 complex consists mainly of GE2270 A (MW 1289), a thiazolyl peptide antibiotic whose structure originates from the modification of a chain of 14 amino acids in a process which creates six thiazole rings and one pyridine. Together with the main component, a number of structurally related molecules are co-produced in small quantities by fermentation. A preparative high-performance liquid chromatrography method was developed to isolate GE2270 factors B1, B2, C1, C2a, C2b, D1, D2, E and T. Their structures, preliminarily determined by 1H-nuclear magnetic resonance spectroscopy in comparison with GE2270 A, were confirmed by low and high resolution fast-atom bombardment mass spectrometry and studies on the intact molecules and on their main hydrolysis products. Their molecular weights range from 1246 to 1306 Da. The structural differences between the factors lie in the extent of methylation and/or oxidation of thiazole rings (D and E) and asparagine, and in the aromatization of the oxazoline ring.

Anti-Bacterial Agents↗
PubMed1994

Use of stable dye-DNA intercalating complexes to detect cystic fibrosis mutations.

Several dyes are now known which intercalate into double-stranded DNA with fluorescence enhancement. One such is TOTO (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl-2,3- dihydro-(benzo-1,3-thiazole)-2-methlyidene]-quinolinium tetraiodide), a homodimer of thiazole orange, whose complexes with DNA are stable to electrophoresis and show a 1000-fold enhancement after binding. We demonstrate that this reagent can be successfully used in combination with a laser-excited fluorescence gel scanner to detect the three most common cystic fibrosis mutant alleles, delta F508, G551D and G542X. This has potential application to mass population heterozygote screening.

Alleles↗
PubMed2000

Rapid and specific detection of PCR products using light-up probes.

Newly developed light-up probes offer an attractive tool for PCR product detection. The light-up probe, which consists of a thiazole orange derivative linked to a peptide nucleic acid oligomer, hybridizes specifically to complementary nucleic acids. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly fluorescent. This eliminates the need to separate bound from unbound probes and reduces the risk of cross contamination during sample handling. We demonstrate here the applicability of light-up probes in two different PCR assays, one directed towards the human beta-actin gene and the other towards the invA gene of Salmonella. The probes do not interfere with the PCR reaction and can either be included in the sample mixture or added after completed amplification. The specificity of the probe is found to be excellent: a single-base mismatch in the target sequence is sufficient to prevent probe binding as indicated by the lack of fluorescence increase. Furthermore, a clear correlation is found between the intensity of gel bands and the measured probe fluorescence in solution, which suggests that the amount of PCR products can be quantified using light-up probes.

Actins↗
PubMed2000

Steric control of DNA interstrand cross-link sites of trans platinum complexes: specificity can be dictated by planar nonleaving groups.

Recent findings that novel trans-dichloroplatinum(II) complexes exhibit antitumor activity violate the classical structure-activity relationships of platinum(II) complexes. These novel "nonclassical" trans platinum complexes also comprise those containing planar aromatic amines. Initial studies have shown that these compounds form a considerable amount of DNA interstrand cross-links (up to approximately 30%) with a rate markedly higher than clinically ineffective transplatin. The present work has shown, using Maxam-Gilbert footprinting, that trans-[PtCl2(NH3)(quinoline)] and trans-[PtCl2(NH3)(thiazole)], representatives of the group of new antitumor trans-dichloroplatinum complexes containing planar amines, preferentially form DNA interstrand cross-links between guanine residues at the 5'-GC-3' sites. Thus, DNA interstrand cross-linking by trans-[PtCl2(NH3)(quinoline)] and trans-[PtCl2(NH3)(thiazole)] is formally equivalent to that by antitumor cisplatin, but different from clinically ineffective transplatin which preferentially forms these adducts between complementary guanine and cytosine residues. This result shows for the first time that simple chemical modification of the structure of an inactive compound alters its DNA binding site into a DNA adduct of an active drug.

Antineoplastic Agents↗
PubMed1985

Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles.

6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.

Catalysis↗
PubMed1995

Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected by Plasmodium falciparum.

The immunofluorescence detection of parasite-specific antigens on the surface of red blood cells infected by Plasmodium falciparum parasites is usually performed by visual detection under a fluorescence microscope. We describe here a technique permitting the analysis of surface immunofluorescence labelling by flow cytometry. Infected red blood cells are selected on the basis of their parasitic DNA and RNA content by Hoechst and Thiazole Orange vital dyes. Cytometric analysis of these labels, as well as general erythrocyte characteristics assessed by analysis of forward and side scatter allows the selection of viable intact infected erythrocytes from other blood cells. The integrity of these selected erythrocytes was confirmed by the absence of labelling with antibodies directed against internal components such as spectrin. This technique permits the detection of specific surface immunofluorescence staining on red blood cells infected with mature stages of P. falciparum by antibodies in sera from hyperimmune Saimiri monkeys. Using Thiazole Orange dye for detection of parasitised cells, this analysis was performed on a FACSscan apparatus equipped with a single laser.

Adult↗