[A multicenter survey of the accessibility of essential medicines for children in China].

Objective: To investigate the availability, prices and affordability of essential medicines in pediatric population across China, in the hope of improving rational use of medicines. Methods: A multicenter cross-sectional survey of medicine prices, availability and affordability was conducted in 17 provinces, municipalities and autonomous region across east, south-central part, west and north of China. Data on 42 medicines used in pediatric population, both original and generic, were collected in 55 public hospitals from May 26 to June 2, 2017. Availability was expressed as the percentage of hospitals with stock of the target medicine on the day of data collection,and median price ratio (MPR) was the ratio of price upon investigation to international reference. Based on national minimum daily wage, affordability represents the number of working days needed to earn the expense which covers a standard course using the target medicine. Statistical software SPSS 13.0 was applied for descriptive analysis of availability, MPR and affordability. Results: Mean Availability of original and generic medicine was 33% and 32%, with median MPR being 5.43 and 1.55. Among the 19 medicines with price information for both original and generic product, the median MPR was 7.73 and 2.04 respectively. Regarding the five medicines used to treat four common pediatric diseases (pneumonia,peptic ulcer, congenital hypothyroidism, refractory nephrotic syndrome), the affordability was 0.63 (0.16-6.17) d for generic medicine, and 1.03 (0.16-11.53) d for its original counterpart. Conclusions: The availability to both original and generic products of the 42 medicines used in pediatric population was low in China. The prices of generic medicines seem to be lower and affordability higher than those of original medicines. There is an urgent need to improve the availability and affordability of pediatric medicines.

Overexpression of SDH confers tolerance to salt and osmotic stress, but decreases ABA sensitivity in Arabidopsis.

Sorbitol dehydrogenase (SDH) catalyses the reversible oxidation of sorbitol, xylitol and ribitol to their corresponding ketoses. In this study, we investigated the expression and role of Arabidopsis SDH in salt and osmotic stress tolerance, and abscisic acid (ABA) response. The expression patterns of SDH were investigated using transgenic Arabidopsis plants expressing beta-glucuronidase (GUS) under control of the promoter with the first intron of SDH. qRT-PCR and histochemical assay of GUS activity were used to study SDH expression regulation by ABA, salt and osmotic stress. SDH-overexpression lines of Arabidopsis were used to investigate the role of SDH in salt and osmotic stress, and ABA response. Arabidopsis SDH was predominantly expressed in source organs such as green cotyledons, fully expanded leaves and sepals, especially in vascular tissues of theses organs. SDH expression was inhibited by NaCl and mannitol treatments. Seed germination and post-germination growth of SDH-overexpressing lines exhibited decreased sensitivity to salt and osmotic stress compared to WT plants. The transcript of SDH was induced by ABA. Overexpression of SDH decreased sensitivity to ABA during seed germination and post-germination growth. Expression of AAO3 increased but ABI5 and MYB2 decreased in SDH-overexpressing lines after ABA treatment. This study demonstrates that expression of SDH is regulated by ABA, salt and osmotic stress. SDH functions in plant tolerance to salt and osmotic stress, and ABA response via specific regulating gene expression of ABA synthesis and signalling in Arabidopsis.

Overexpression of forkhead box protein M1 (FOXM1) plays a critical role in colorectal cancer.

BACKGROUND: The forkhead box M1 (FOXM1), an important regulator of cell differentiation and proliferation, is overexpressed in a number of aggressive human carcinomas. However, the clinical significance of FOXM1 signaling in human colorectal cancer (CRC) pathogenesis remains unknown. The aim of this study was to evaluate the role of FOXM1 in CRC tumorigenesis. METHODS: We investigated FOXM1 expression in 103 cases of primary CRC and matched normal tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on CRC proliferation and metastasis using in vitro models of CRC. RESULTS: The results showed that high expression of FOXM1 staining was 85.44% (88/103) in 103 cases of CRC and 20.39% (21/103) in 103 cases of adjacent non-cancerous tissue samples; the difference of FOXM1 expression between two groups was statistically significant (P < 0.001). Silencing of FOXM1 inhibited the proliferation of CRC cells, and the invasion and migration of CRC cells were distinctly suppressed. Furthermore, FOXM1 knockdown led to substantial reductions in VEGF-A levels in CRC cell lines. CONCLUSIONS: Our data suggest that the pathogenesis of CRC maybe mediated by FOXM1, and FOXM1 could represent selective targets for the molecularly targeted treatments of CRC.

Interactions between adenovirus E1A and members of the AP-1 family of cellular transcription factors.

We have studied interactions between bacterially produced E1A linked to Sepharose and the various DNA-binding proteins present in HeLa cell nuclear extracts (NE). DNA-binding activities and cross-reactive polypeptides recognizing the cAMP-responsive element (CRE) and the activator protein 1 (AP1) sites were bound to the E1A column, whereas nuclear factor 1 (NF1) and the activator protein 2 (AP2) DNA-binding activities were not retained by E1A. The binding activities that were retained belonged to the CRE and JUN protein family, as judged by Western blot analysis. Authentic CRE-BP1, c-Jun and c-Fos proteins produced by in-vitro translation also bound to the E1A column. However, efficient binding of in-vitro-translated CRE-BP1 and c-Fos proteins to E1A required preincubation with NE. We show here that immobilized E1A sequesters several cellular upstream transcription activators, and suggest a role for members of the AP1 family of transcription factors in E1A-mediated gene regulation.

Effects of N1-guanyl-1,7-diaminoheptane, an inhibitor of deoxyhypusine synthase, on the growth of tumorigenic cell lines in culture.

N1-guanyl-1,7-diaminoheptane (GC7) is a potent inhibitor of deoxyhypusine synthase (DHS), the enzyme that catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF-5A). Since eIF-5A is the only known cellular substrate for DHS and GC7 has been reported to block the proliferation of CHO cells, it has been suggested that DHS may be a novel target for anti-cancer therapy. In the present study we investigated the antiproliferative effect of GC7 on several tumorigenic cell lines under various growth conditions. We found that this compound inhibits the proliferation of H9 cells in suspension culture and the growth of HeLa cells and v-src-transformed NIH3T3 cells under both anchorage-dependent and anchorage-independent conditions. Moreover, studies with NIH3T3 cells and v-src-transformed NIH3T3 cells show that GC7 inhibits the growth of both cell lines in monolayer culture with similar potency and could not reverse the transformed phenotype. In addition, the v-src-transformed cells grown under both anchorage-dependent and anchorage-independent conditions showed similar sensitivity toward GC7. These data indicate that GC7 acts as a general antiproliferative agent and does not appear to preferentially target tumorigenic cell types. Cell cycle analysis show that GC7 reduces the CHO-K1 cell population in the G1-phase of the cell cycle by 42% and increases the number of cells in the S-phase by 44%. This cell cycle distribution profile strikingly resembles the distribution of cells treated with puromycin. This result supports the hypothesis that the synthesis of a subset of proteins important for the S-phase progression of CHO-K1 cells might be dependent upon hypusinated eIF-5A. Thus the antiproliferative effect of GC7 appears to be related to its interference with the progression of cell cycle, which also provides a possible explanation for the lack of selectivity of GC7 between nontransformed and transformed cell types tested in this study.

Adreno-cholinergic modulation of junctional communications between the pigmented and nonpigmented layers of the ciliary body epithelium.

PURPOSE: Cell-to-cell communications between the epithelial layers of the ciliary body may be critical for aqueous humor production. The aim of this study was to identify pharmacologic agents that affect this path. METHODS: Whole New Zealand rabbit ciliary bodies were mounted in Ussing-type chambers with Ca2+(-)free and Ca2+(-)rich Tyrode's in the nonpigmented (NPE; aqueous) and pigmented (PE; serosa) epithelial side hemichambers, respectively. The NPE of the PE were then permeabilized, either selectively to monovalent ions with amphotericin B or nonselectively to small solutes with digitonin. Resultant active transport activities were tracked as short circuit currents (ISCS). RESULTS: Permeabilization of the NPE with either 10 microM amphotericin B or 10 micro M digitonin led to an aqueous-to-serosa-positive ISC. This ISC was inhibited by serosal-side ouabain and heptanol, indicating movement of Na+ from permeabilized NPE to the PE by the interlayer junctional path, followed by PE-to-serosa active Na+ transport. Permeabilization of the PE with amphotericin B elicited an ISC in the opposite direction, This ISC was abolished by aqueous-side ouabain and by heptanol, consistent with sequential PE to NPE Na+ translocation, followed by active, NPE-to-aqueous transport. Acetylcholine, epinephrine, norepinephrine, and the alpha 1-adrenergic agonist phenylephrine, but not brominidine, an alpha 2-adrenergic agonist, each caused an approximately 50% reduction of these currents. The inhibitions were fully dependent on serosal-side Ca2+ and were blocked by one calmodulin inhibitor, trifluoperazine, but not by another, calmidazolium. CONCLUSIONS: The above observations provide evidence that cholinergic or alpha 1-adrenergic activation of the PE causes Ca2+(-)dependent inhibition of the NPE-PE junctional path. A triflouperazine-sensitive entity, which may be distinct from calmodulin, is involved in the inhibition.

Human erythroleukemic (HEL) cells express a platelet P2T-like ADP receptor.

Treatment of Fura-2 loaded HEL cells, a human megakaryocyte-like cell line, with P2-purinoceptor nucleotide ligands (ADP, ATP, UTP, 2-methylthio-ATP) evoked a rise in cytosolic calcium. Homologous- and cross-desensitization studies using sequential addition of nucleotides showed that the ADP-induced calcium response in HEL cells was mediated mainly by purinoceptor(s) for which ADP but not ATP was an agonist. There were also minor contributions from purinoceptors for which ATP and ADP are both agonists (probably P2U and P2Y). ATP inhibited the ADP-induced calcium response in HEL cells. This inhibition was overcome by raising the ADP concentration, thus indicating that ATP was an antagonist for the HEL cell ADP receptor. AMP was also an antagonist, albeit weak, for the HEL cell ADP receptor. Antagonism of the ADP-induced calcium response by ATP was similarly observed in MEG-01 cells, another human megakaryocyte-like cell line, but not in 293 cells, a nonhematopoietic cell line. These studies suggest that HEL cells express an ADP receptor for which ATP and AMP are antagonists. This characteristic of the HEL cell ADP receptor is also displayed by the platelet P2T receptor. Thus, HEL cells appear to be a surrogate source of the platelet ADP receptor.

Active sodium and chloride transport across the isolated rabbit conjunctiva.

The bulbar-palpebral conjunctiva from albino rabbits was dissected as a cylinder and cut longitudinally to convert it to a flat epithelium that was mounted as a partition between Using-type chambers, exposing 0.38 cm2 of cross-sectional area. The tissue was bathed with a modified Tyrode's solution at 37 degrees C, pH 7.5. The tear-facing side (apical) was 14.6 +/- 1.5 mV negative relative to the basolateral side. Transepithelial resistance was 1.23 +/- 0.01 K omega.cm2 and the short-circuit current (Isc) was 14.4 +/- 1.3, microA/cm2. Sixty percent of the Isc could be accounted for by a Na(+)-dependent, bumetanide-inhibitable Cl- transport directed towards the apical side. The remainder of the Isc reflected a Na+ absorptive process at the apical surface that was amiloride resistant. Evidence was obtained that a likely contributor to this activity is an electrogenic Na(+)-glucose co-carrier. The Cl-dependent Isc was stimulated by forskolin and epinephrine. Permeabilization of the apical membrane with amphotericin B evinced a current carried by a basolateral Na+:K+ pump. An effect by heptanol suggested that part of the Isc traverse the epithelium via gap junctions. Our results imply that transport processes at the conjunctiva could influence the composition of the tear film.

Ascorbate-stimulated active Na+ transport in rabbit ciliary epithelium.

In a physiological medium (134 mM Na+ concentration), unidirectional blood-to-aqueous and aqueous-to-blood Na+ fluxes across the isolated rabbit ciliary epithelium are large, rendering the detection of a net transport difficult. At 134 mM an active component for Na+ may be obscured by diffusional fluxes and a bidirectional Na(+)-Cl- cotransport. Considering that the active transport saturates at about 30 mM, experiments were performed at this reduced Na+ concentration to minimize the influence of diffusional pathways. A net blood-to-aqueous Na+ flux that ranged from 0.25 to 0.81 mu eq/hr was obtained. Addition of ascorbic acid to the aqueous side under this condition increased the blood-to-aqueous flux with little effect on the flux in the opposite direction. Ouabain inhibited both the Na+ and ascorbate-stimulated Na+ transport. The increase in blood-to-aqueous Na+ flux by ascorbate was also observed in tissues bathed with [Na+] closer to physiological levels (100 mM). These results indicate that the rabbit ciliary epithelium transports Na+ into the posterior chamber. Since aqueous ascorbate stimulates Na+ transport, it may be implicated in both Na+ movement and aqueous humor secretion. However, the rate of Na+ transport can only account for a small fraction of total aqueous humor production.

Slow light and dark adaptation of horizontal cells in the Xenopus retina: a role for endogenous dopamine.

A role for endogenous dopamine in the control of rod and cone contributions to a second-order retinal neuron, the horizontal cell (HC) was studied in the Xenopus retina. Relative rod and cone contributions were estimated from HC responses to scotopically balanced 491- and 650-nm flashes. In eyecups prepared in light then placed in darkness, cone input to the HC slowed and diminished on a time scale of hours. The decline in cone input was balanced by a slow growth of rod input to the HC. Administration of D-amphetamine, a dopamine releasing agent, restored the light-adapted waveform. The kinetics of slow light adaptation were examined by recording HC responses from eyecups that had been dark-adapted previously for 11-14 h. When test flashes fell on a dark field, cone input to the HC grew for 2-4 h, reached a plateau, and later declined. If, however, flashes were superimposed on a weak background field, cone input to the HC continued to increase monotonically at about 10%/h. This increase was abolished by superfusion with a nonspecific dopamine receptor blocker, cis-flupenthixol (50 microM), resulting in the complete suppression of cone-to-horizontal cell synaptic transfer and the enhancement of rod-to-horizontal cell communication. Subcutaneous injection of reserpine, a drug that depletes dopamine stores (2 mg/kg on 1-4 successive days), or intraocular injection of the dopamine neurotoxin, 6-hydroxydopamine (10-30 micrograms) slowed and reduced the amplitude of cone input to the HC, even in completely light-adapted eyes.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein factor(s) binding independently to two different regions of the adenovirus 2 major late promoter.

The protein factor(s) in a fraction from the HeLa cell nuclear extract required for specific in vitro transcription can specifically bind to adenovirus 2 major late promoter (Ad 2 MLP) DNA. We demonstrate by in vitro footprinting assay that there are two asymmetric protected regions covering the TATA box and the nucleotides upstream from the TATA box. In the coding strand, the DNAse I protected regions span from nucleotides -10 to -50 and from -52 to -68. In the noncoding strand, the protected regions span from nucleotides -10 to -32 and from -45 to -65. Using different Ad 2 MLP point mutants in this assay, we show that the transcriptional down mutants of the TATA box (AC-30 and AC-28) abolish the binding of protein factor(s) to the TATA box but do not affect binding in the upstream region. The new upstream transcriptional down mutant (TA-56) abolishes the binding of protein factor(s) in the upstream region but does not affect binding to the TATA box. The mutants which do not affect transcription efficiency (GA-51 and CG-61) do not modify the binding to either the TATA box or the upstream region. Methylation protection experiments show that the guanosines at -58 and -60 in the coding strand and at -57 (probably also -55) in the noncoding strand are in close contact with protein factor(s). The results indicate that the TATA box and its upstream region of Ad 2 MLP are independently bound by at least two different factors in vitro.

Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein.

The subcellular distributions of the endogenous eukaryotic translation initiation factor, eIF-5A, and Rev, a protein of the human immunodeficiency virus proposed to interact with eIF-5A, were studied in COS-7 cells treated with inhibitors of RNA or protein synthesis. We have previously shown that transiently expressed Rev is localized in the nucleolus, whereas eIF-5A is primarily in the cytoplasm. The subcellular localization of Rev was not affected by treatment with protein synthesis inhibitors (cycloheximide, CHX, 10 micrograms/ml; puromycin, 10 micrograms/ml), although its location changed from predominantly the nucleolus to the cytoplasm after treatment with RNA synthesis inhibitors (actinomycin D, 4 micrograms/ml, and 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole, DRB; 0.1 mM), as previously reported. In contrast, none of the RNA synthesis inhibitors (alpha-amanitin, 10 micrograms/ml; actinomycin D, 4 micrograms/ml, and DRB, 0.1 mM) caused any significant changes in the subcellular distribution pattern of eIF-5A. However, treatment with puromycin, a protein synthesis inhibitor known to dissociate ribosomes, dramatically altered the subcellular distribution pattern of eIF-5A in 30% of the cell population. In these cells, the staining of eIF-5A was changed from an endoplasmic reticulum (ER) net work-like perinuclear structure to a patched dotted pattern dispersed throughout the cytoplasm. This change was not observed in the same cells stained for calnexin, an ER resident protein, nor in cells treated with CHX, which freezes the ribosomes to block protein synthesis. Our data suggest that eIF-5A does not shuttle between the nucleus and cytoplasm in the same way as Rev. Our findings are consistent with our previous conclusion that eIF-5A is associated with the ER through ribosomes and support a role for eIF-5A in protein synthesis.

Reduction in water permeability of the rabbit conjunctival epithelium by hypotonicity.

The effects of unilateral exposure to hypotonic media on the diffusional water permeability of the isolated rabbit conjunctiva were determined. For these experiments, a segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuited conditions. Unidirectional diffusional water fluxes (Jdw) were measured in either direction by adding 3H2O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. Jdw were determined in control isosmotic conditions and after dilution of one of the bathing solutions from 290 to 108 mOsMolar. This hypotonic condition reduced Jdw by 25-30% (n = 17) when applied basolaterally and by 25% (n = 6) apically. The effects were reversible and were also obtained when the opposite bathing solution contained amphotericin B, selectively permeabilizing the contralateral cell surface. From concomitant changes in transepithelial electrical resistance as well as 14C-mannitol fluxes completed under identical conditions, arguments are presented that the above effect is best explained as a cell regulated reduction in membrane water permeability. Presumably both apical and basolateral membranes can down-regulate their water permeabilities. This response, suggesting a protective mechanism to help maintain cell volume from hypotonicity, was also seen in other studies using the amphibian bladder and the frog cornea, in which the effect was only obtained basolaterally. Thus, regulation of epithelial water permeability appears to be a basic trait common to both amphibians and mammals, although tissue differences exist.

The subcellular distribution of eukaryotic translation initiation factor, eIF-5A, in cultured cells.

To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus oocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.

Faithful transcription of ribosomal 5-S RNA in vitro depends on the presence of several factors.

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5-S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G-100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5-S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5-S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 +/- 1 kDa stimulates the formation of 5-S RNA. This protein is contained in the breakthrough of DEAE-cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl. In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G-100 contains functional complexes, which are capable of transcribing the cloned 5-S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio-Gel A5m.

Isolation of a transcription complex for ribosomal 5S RNA.

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non-denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re-initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA-binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.

Effect of heptanol on the short circuit currents of cornea and ciliary body demonstrates rate limiting role of heterocellular gap junctions in active ciliary body transport.

Rabbit ciliary body and cornea were mounted in Ussing-type chambers in Tyrode's under voltage clamp and the effects of heptanol, a gap junction inhibitor, on the short circuit current generated by each of the respective epithelia were determined. Studies were carried out either in control conditions or following amphotericin B permeabilization of either the basolateral membrane of the nonpigmented epithelium of the ciliary body or the apical membrane of the corneal epithelium, respectively. Previous studies have shown that, following these permeabilizations, short circuit currents are established, reflecting aqueous (or tear)-to-serosa Na+ fluxes, and that Na+ translocation through gap junctions connecting the individual layers of these tissues constitutes the major rate limiting step. Heptanol inhibited most of the short circuit current of the amphotericin B-modified ciliary body and cornea and of the unmodified ciliary body epithelium (control). In all these cases, the apparent IC50 was about 0.8 M. In the unmodified corneal epithelium, where ion translocation across the apical membrane constitutes the main rate limiting step for active secretion, 0.4 or 0.8 mM heptanol induced short circuit current increases; partial inhibition was observed only at high concentrations known to cause maximal inhibition of junctional permeability. Heptanol also enhanced the volume regulatory decrease of cultured human NPE cells, a process dependent on cell swelling-induced stimulation of Cl- and K+ permeabilities. Combined with our previous results demonstrating the lack of heptanol effects on other epithelial functions, these data suggest that the effect of heptanol on the active ciliary body transepithelial transport is primarily due to inhibition of the nonpigmented-pigmented junctional path and that this path is a potential site of rate limitation for the secretory process.

Targeted delivery of peptide epitopes to class I major histocompatibility molecules by a modified Pseudomonas exotoxin.

Cytotoxic T lymphocytes (CTLs) expressing the CD8 surface marker recognize peptides in association with major histocompatibility complex (MHC) class I molecules. Although most peptides expressed on MHC class I molecules are derived from self- or virally encoded proteins, delivery of exogenous proteins to the cytosol can result in their being processed for presentation to CTLs on MHC class I molecules. We describe two fusion proteins (PEMa and PENP), consisting of the binding and translocating domains of Pseudomonas exotoxin A (PE), fused to peptide epitopes from influenza A matrix protein and nucleoprotein, respectively. These fusion proteins were internalized and processed by MHC class I-positive target cells, resulting in sensitization of target cells for lysis by peptide-specific CTLs. A point mutation known to interfere with intoxication by wild-type PE also reduced the ability of PEMa to sensitize target cells. Fusion of peptide or polypeptide epitopes with PE provides a potential means of eliciting CTLs without the use of self-replicating agents, as well as a useful probe for studying MHC class I-restricted antigen processing.