Insertion of tear proteins into a meibomian lipids film.

The eyelid meibomian gland secretions form the outer layer of the tear film. That layer functions as a lubricant during a blink, and as a barrier against intrusion of foreign bodies. The lipid film is also exposed to proteins present in the aqueous phase that may adsorb there, and thus form an integral part of the surface of the tear film, or possibly, cause disruption to the outermost layer. Therefore, the adsorption of tear proteins to the meibomian lipid layer was object of the present investigation. A model tear was set up coating a pendant drop of saline with a film of meibomian lipids and measuring variations of the interfacial pressure after the injection of tear proteins into the aqueous subphase at their physiological concentration. All tear proteins adsorbed at the interface causing the initial surface pressure to increase. For each protein, a limiting surface pressure at which a given protein was no longer able to insert into the lipid layer was found. Among the proteins tested, lipocalin was the most surface active one and inserted into the lipid layer in the whole range of surface pressure exerted by the meibomian lipid mixture. Lactoferrin, lysozyme and IgA also interacted with the lipids whereas albumin interacted more weakly. The timescale of the protein insertion into the lipid layer was of the order of 10(2) s. It was hypothesized that protein adsorption at the interface could be associated with structural changes. Indeed, the enzymatic activity of lysozyme was maintained in the presence of an outermost meibomian lipid layer that prevented its denaturation while exposure at the air/aqueous interface induced significant lysozime degradation. meibomian lipid composition is therefore functional to maintain tear proteins activity.

Cytokeratin expression in primary epithelial cell culture from bovine conjunctiva.

Aim of the present study is to extend our previous observations on a model of primary epithelial cell culture obtained from bovine conjunctiva, and analyse the maintenance of the conjunctival phenotype, relative to cytokeratin (CK) expression, through extended periods of cultivation under different conditions. Conjunctival epithelial cells were grown in transwell filters, and cultured either under liquid covered (LC), or air-interface (AI) conditions. The physiological state of the cells was monitored daily by measurement of the trans-epithelial electrical resistance (TEER). Analysis of cytokeratin expression was then carried out at different time points (up until 14 days), and compared to the original profile of the conjunctival tissue in order to assess deviations from the primitive phenotype. Immunodetection studies, carried out by both western immunoblot and immunofluorescence analyses, revealed constant expression of the pan-epithelial marker AE3 (recognizing basic type cytokeratins), confirming the epithelial nature of the culture. Other cytokeratins characteristic of non-keratinized stratified epithelia (CK4 and CK13) were absent in corneal tissue, while in conjunctival epithelial cells were more expressed under AI than under LC culture conditions. Expression of CK12, a specific marker of corneal tissue, revealed by the antibody AE5, was never observed in conjunctival epithelial cells. These results indicate that the conjunctival phenotype is conserved during extended periods of culturing, making this system a reliable substitute of conjunctival tissue for pharmaceutical analyses.

Identification of a developmentally regulated transcript expressed during encystation of Entamoeba invadens.

Differentiation of trophozoites into cysts in Entamoeba species has been described morphologically and to a lesser extent biochemically, but studies of stage specific gene expression have not been reported. At present Entamoeba invadens is the only species that can be induced to differentiate in axenic culture and is a useful model system for the human parasite Entamoeba histolytica. Using this model system, we performed cDNA-mRNA hybridization experiments to compare the RNA populations from trophozoites and from parasites at different stages of cyst formation. We detected the accumulation of a population of stage specific transcripts between 8 and 22 h after parasites are placed in induction medium. To identify genes involved in the trophozoite-cyst transformation we carried out a differential screening of a cDNA library. This yielded a clone that represents a stage specific gene whose transcripts are barely detectable in vegetatively grown trophozoites and maturing cysts, but are readily detected at the onset of cyst formation. Other features of the gene and its predicted protein product(s) are described.

Increased levels of polyadenylated histone H2B mRNA accumulate during Entamoeba invadens cyst formation.

We have isolated cDNA clones of a member of the histone H2B protein family by differential screening of an Entamoeba invadens cDNA library with cDNA probes from vegetatively growing or encysting parasites. The cDNA clones reveal two polyadenylation sites, 26 nucleotides and 31 nucleotides downstream from the stop codon. RNA species recognized by E. invadens histone H2B clones are found at increased levels during cyst formation. Histone H2B RNA could be detected in both the poly(A)+ and poly(A)- RNA fractions, with stage-specific differences in the steady state levels of the two RNAs: trophozoites contain predominantly the poly(A)- RNA, while encysting parasites express predominantly the poly(A)+ RNA. Southern blot analysis suggests that both forms are transcribed from a single copy gene.

The small ribosomal subunit RNA isoforms in Plasmodium cynomolgi.

We report the isolation, characterization and analysis of the small subunit rRNA genes in Plasmodium cynomolgi (Ceylon). As in other Plasmodium species, these genes are present in low copy number, are unlinked and form two types that are distinct in sequence and are expressed stage specifically. The asexually expressed (type A) genes are present in four copies in the Ceylon- and in five copies in the Berok-strain. Surprisingly, the sexually expressed (type B) gene is present in a single copy. The vast majority of the differences between gene types is confined to the variable regions. The pattern of divergence is different from that observed in Plasmodium berghei or in Plasmodium falciparum. Analysis of the small subunit rRNA sequences of P. cynomolgi, P. berghei and P. falciparum, indicates that the two gene types do not evolve independently but rather interact (through gene conversion or some form of recombination) to such an extent as to erase whatever stage-specific sequence signatures they may have had in the last common ancestor.

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.

The evolution of plasmodial stage-specific rRNA genes is dominated by gene conversion.

Plasmodium species exhibit the unprecedented situation of distinct, stage-specific rRNA sequences. We present an analysis of two pairs of sequences of the small rRNA subunit (Plasmodium falciparum and Plasmodium berghei) and show that these genes do not evolve independently and that in fact their evolution is dominated by gene conversion. This analysis also shows that no extensive stage-specific sequences are conserved in the two species, thus rendering unlikely that the existence of stage-specific rRNA genes results from a requirement for distinct rRNA types.

Plasmodium yoelii nigeriensis circumsporozoite gene structure and its implications for the evolution of the repeat regions.

The circumsporozoite (CS) gene encodes the most immunogenic component of the plasmodial sporozoites. The immunodominant epitope-encoding domain of the CS gene shows sequences that are repeated in tandem. A detailed analysis of the CS repeats of certain closely related malaria parasites (strains of Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium vivax) showed that they evolve rapidly yet are well conserved within the gene. We were interested in studying whether the CS repeats of Plasmodia more distantly related to these species evolve in a similar manner. To this end, we isolated and characterized the Plasmodium yoelii nigeriensis CS gene. A comparative analysis of its sequence with that of Plasmodium yoelii yoelii shows that both have three sets of repeats, termed PR, R1, and R2. The R1 and basically also the R2 sequences show the features observed in most CS repeats, i.e., they evolve rapidly and are nearly perfectly tandemly repeated. In contrast, the PR repeats are not internally conserved nor divergent in sequence. The implications of these findings for the evolution of the CS repeats are discussed.

Plasmodium falciparum: restricted polymorphism of T cell epitopes of the circumsporozoite protein in Brazil.

We examined the extent of variation of the 3' region of the circumsporozoite gene among Plasmodium falciparum isolates through amplification of a selected DNA fragment followed by DNA sequencing. A total of 32 isolates were analyzed, of which 24 were from Amazon endemic areas in Brazil and 8 from widely separated geographical regions in the world. Among Brazilian isolates only 2 variants were detected: 19 displayed the same sequence of strain 7G8 whereas the 4 remaining isolates differed from the 7G8 strain at five nucleotide positions which also led to amino acid changes. Variation was restricted to one of the T-helper epitopes while the sequence identified as a cytotoxic T cell epitope was conserved in all Brazilian isolates. P. falciparum samples from other geographical regions in the world showed sequences distinct from those of Brazilian isolates. However, some constancy could be observed within that variation. For instance, the most frequent nucleotide substitutions, from A and C at nucleotide positions 1015 and 1024, were the same in all isolates.

On the evolutionary history of the circumsporozoite protein in plasmodia.

We report the complete nucleotide sequence of the circumsporozoite (CS) gene of Plasmodium brasilianum and present an analysis of its evolutionary profile. Despite the lack of a reliable time scale, the analysis of the number and distribution of fixations among seven taxa provides a first glimpse of the evolutionary history of the CS gene, and suggests that the branching events of this gene are completely unconnected with--and far precede in time--the speciation event of the parasite's vertebrate hosts.

Circumsporozoite gene of a Plasmodium falciparum strain from Thailand.

The nucleotide and deduced amino acid sequences of the CS gene of a Plasmodium falciparum strain from Thailand (T4) are presented. Comparison with the nucleotide sequences of two other P. falciparum CS genes, 7G8 from Brazil and Wellcome from West Africa, shows that: the coding regions outside the repeats of T4 and 7G8 are co-extensive and lack 30 nucleotides present in the Wellcome strain 5' to the repeats; in this region, T4 also differs at 3 nucleotide positions from the 7G8 and the Wellcome strains; in the region 3' to the repeats, T4 differs at two positions from 7G8 and at two other positions from the Wellcome strain--remarkably, all of these differences result in amino acid substitutions; the structure of the tandem repeats in the CS gene of T4 is, 5' to 3', [NANP-NVDP] X 3, [NANP] X 38, which is different from that of the two other strains. Due to the use of synonymous codons, the repetition of the sequence is more precise at the amino acid level than at the nucleotide level. These features contrast with those observed in the CS genes of other plasmodial species.

Expression in yeast of a Plasmodium vivax antigen of potential use in a human malaria vaccine.

DNA coding for 234 amino acids of the circumsporozoite (CS) protein of Plasmodium vivax was incorporated into yeast expression vectors. The DNA encoded all the repeat domain and codons for a highly conserved sequence, KLKQP, found in CS proteins from all malaria parasites. Yeast cells transformed with these autonomously replicating plasmids expressed, upon induction, high levels of the CS polypeptide. The malaria antigen was purified in good yields from yeast extracts and was injected into mice using alum as adjuvant. The antibodies recognized the authentic CS protein, and at high dilutions, they inhibited the invasion of hepatocytes by sporozoites in vitro.

The circumsporozoite gene of the Plasmodium cynomolgi complex.

An analysis of the circumsporozoite (CS) genes of six closely related plasmodia is presented. Like other plasmodial antigens, the CS protein contains tandem repeats flanked by conventional nonrepeated sequences. Our analysis shows that the repeats, which encode the immunodominant epitope of the CS protein, diverge more rapidly than the remainder of the gene, and that the maintenance and evolution of the repeats cannot be explained as the result of selection at the protein level. We argue that a mechanism acts directly on the DNA sequence to constrain the internal divergence of the repeats, and as a result promotes their rapid divergence between taxa.

Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.

The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes.

Inhibition of development of exoerythrocytic forms of malaria parasites by gamma-interferon.

A specific DNA probe was used to study the effect of recombinant rat, mouse, and human gamma-interferon (gamma-IFN) on the course of sporozoite-induced malaria infections. In mice and rats infected with sporozoites of Plasmodium berghei, mouse and rat gamma-IFN's strongly inhibited the development of the exoerythrocytic forms in the liver liver cells of the hosts, but not the development of the erythrocytic stages. The degree of inhibition of the exoerythrocytic forms was proportional to the dose of gamma-IFN administered, but was independent of the number of sporozoites used for challenge. A 30 percent reduction in the development of exoerythrocytic forms in rat liver was achieved when 150 units (about 15 nanograms of protein) of rat gamma-IFN were injected a few hours before sporozoite challenge; the reduction was 90 percent or more with higher doses of gamma-IFN. The effect was less pronounced if the gamma-IFN was administered 18 hours before or a few hours after challenge. Human gamma-IFN also diminished the parasitemia in chimpanzees infected with sporozoites of the human malaria parasite Plasmodium vivax. The target of gamma-IFN activity may be the infected hepatocytes themselves, as shown by in vitro experiments in which small doses of the human lymphokine inhibited the development of exoerythrocytic forms of Plasmodium berghei in a human hepatoma cell line. These results suggest that immunologically induced interferon may be involved in controlling malaria infection under natural conditions.

Infectivity of Plasmodium berghei sporozoites measured with a DNA probe.

A 2.3 kb, 32P-labeled repetitive DNA probe of Plasmodium berghei was used to measure the amount of parasite DNA in the liver of Norway Brown rats and mice infected with sporozoites. Standard hybridization curves were obtained by probing different amounts (100 pg to 1 microgram) of P. berghei DNA immobilized on nitrocellulose filters. Host DNA did not interfere with hybridization specificity and sensitivity. A 100-fold increase in hepatic parasite DNA was detected between 25 h post-infection and the peak of parasite proliferation, detected at 44 h. The amount of parasite DNA increased with the number of injected sporozoites. At 5 h post-infection, a large proportion of parasite DNA was found in the spleen. However, this diminished with time and was negligible in amount at 25 h. A significant number of viable sporozoites were probably cleared in the spleen, since considerably more parasite DNA was found in the livers of splenectomized rats than in sham-operated counterparts. Although older rats develop much lower parasitemias upon inoculation of sporozoites, no significant differences were observed in the amount of parasite DNA in rats, 43 and 152 days old, injected with equal numbers of sporozoites. The higher resistance to malaria displayed by older rats is probably controlled by post-hepatic events. The infectivity of sporozoites for A/J mice was calculated to be about 1/20th that of Norway Brown rats.

Evolutionary profile of the circumsporozoite gene of the Plasmodium cynomolgi complex.

The circumsporozoite genes and flanking sequences of the Ceylon, Gombak, London, NIH and Mulligan strains of the Plasmodium cynomolgi complex were isolated by molecular cloning and compared. About 11,000 bases of the Gombak clone were mapped in detail and found to have their exact counterparts in all the other strains. In contrast the epitope-encoding region, a 600-base sequence consisting of short tandem repeats, exhibited no homology with any of the other clones. These findings show that different regions of the circumsporozoite gene evolve in sharply different modes.

Sensitive and specific DNA probe for detection of Plasmodium falciparum.

The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences.