Plasticity of heart rate signalling and complexity with exercise training in obese individuals with and without type 2 diabetes.

OBJECTIVE: To examine the responsiveness of cardiac autonomic function and baroreflex sensitivity (BRS) to exercise training in obese individuals without (OB) and with type 2 diabetes (ObT2D). DESIGN: Subjects were tested in the supine position and in response to a sympathetic challenge before and after a 16-week aerobic training program. All testing was conducted in the morning following a 12-h fast. SUBJECTS: A total of 34 OB and 22 ObT2D men and women (40-60 years of age) were studied. MEASUREMENTS: Heart rate variability (HRV) was measured at rest via continuous ECG (spectral analysis with the autoregressive approach) and in response to upright tilt. The dynamics of heart rate complexity were analyzed with sample entropy and Lempel-Ziv entropy, and BRS was determined via the sequence technique. Subjects were aerobically trained 4 times per week for 30-45 min for 16 weeks. RESULTS: Resting HR decreased and total power (lnTP, ms(2)) of HRV increased in response to exercise training (P<0.05). High frequency power (lnHF) increased in OB subjects but not in OBT2D, and no changes occurred in ln low frequency/HF power with training. Upright tilt decreased lnTP and lnHF and increased LF/HF (P<0.01) but there were no group differences in the magnitude of these changes nor were they altered with training in either group. Tilt also decreased complexity (sample entropy and Lempel-Ziv entropy; P<0.001), but there was no group or training effect on complexity. BRS decreased with upright tilt (P<0.01) but did not change with training. Compared to OB subjects the ObT2D had less tilt-induced changes in BRS. CONCLUSION: Exercise training improved HRV and parasympathetic modulation (lnHF) in OB subjects but not in ObT2D, indicating plasticity in the autonomic nervous system in response to this weight-neutral exercise program only in the absence of diabetes. HR complexity and BRS were not altered by 16 weeks of training in either OB or ObT2D individuals.

Adenoviral vectors for in vivo gene delivery to oligodendrocytes: transgene expression and cytopathic consequences.

Replication defective viral vectors provide a potentially useful means of gene transfer to oligodendrocytes and thus for studying the pathogenesis of white matter disease. In this study we have examined the expression pattern of E1/E3 deleted adenoviral vectors expressing the reporter gene LacZ (AdlacZ) as a means of establishing the value of these vectors for gene delivery to oligodendrocytes in adult rat white matter. Our results indicate that although such an approach can be used to induce transgene expression in oligodendrocytes, it is complicated by both immunogenic and cytopathic effects. Thus, in normal animals, injection of DeltaE1/E3 adenoviral vectors was associated with a robust immune response that led to a lack of expression by 40 days after injection. In order to overcome this complication, virus was injected into the white matter of immuno-deficient athymic rats. These experiments indi- cated that even in the absence of a T cell response high viral titres of DeltaE1/E3 adenoviral vectors had a profound cytopathic effect leading to death of oligodendrocytes and hence demyelination. A similar cytopathic effect was demonstrated using an adenoviral vector expressing the neurocytokine ciliary neurotrophic factor (AdCNTF). As the titre of injected virus was decreased there was a significant decrease in the number of transgene expressing cells. These experiments therefore indicated that in immunodeficient recipients there is a narrow window of virus titre that results in a high rate of infectivity and expression without significant cytopathic consequences. At higher viral titres the cytopathic effects include oligodendrocyte death and demyelination, while at lower titres there is a significant decrease in the efficiency of the number of cells expressing the transgene.

Characterization of a mitogen-activated protein (MAP) kinase from Plasmodium falciparum.

A mitogen-activated protein (MAP) kinase gene, PfMAP, from Plasmodium falciparum was recently identified. We expressed this gene in Escherichia coli to test whether it encodes a functional MAP kinase. Recombinant PfMAP kinase autophosphorylates on both the tyrosine and threonine residues within the TXY motif, and readily phosphorylates myelin basic protein as exogenous substrate. This identifies the PfMAP gene product as a true member of the growing family of MAP kinases. Wild-type PfMAP kinase expressed in COS-7 (SV40 transformed African green monkey kidney) cells seemed to induce apoptosis in these cells. Western blots and immunoprecipitations indicated that the kinase is expressed during the growth of the parasite in the red blood cell as three major forms: truncated forms with apparent molecular masses of 40 kDa and 80 kDa, and as a protein of approximately 150 kDa. The 40 kDa form is present throughout the intraerythrocytic development, whereas the two larger forms are only detected in mature parasites. The 40 kDa and 80 kDa forms are tyrosine phosphorylated, indicating that they represent the active forms of the PfMAP kinase. The total PfMAP kinase activity constantly increases with the maturation of the parasite.

Molecular cloning, characterization and localization of PfPK4, an eIF-2alpha kinase-related enzyme from the malarial parasite Plasmodium falciparum.

PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.

Plasmodium falciparum protein kinase 5 and the malarial nuclear division cycles.

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

Molecular cloning and characterization of a putative glutathione reductase gene, the PfGR2 gene, from Plasmodium falciparum.

Recently, glutathione reductase (GR) has emerged as a promising target for antiparasitic drugs. The central role of GR in cellular antioxidant defence, the particular susceptibility of intracellular parasites like Plasmodium falciparum to oxidative stress, and successful inhibitor studies substantiate this approach. However, more information is required on the structural and functional characteristics of GR from malarial parasites and differences from the enzyme of host erythrocytes. We have identified a putative P. falciparum GR gene coding for a polypeptide (PfGR2) of 500 amino acids that exhibits 40-45% sequence identity with GR enzymes from other species. 18 out of 19 residues contributing to glutathione binding are identical in the putative PfGR2 and human GR. According to Southern blot analysis, the PfGR2 gene is present as a single-copy gene. It is expressed during the intraerythrocytic life cycle. Stage-specific Northern blot analysis demonstrates that the PfGR2 gene is only weakly transcribed in ring, early trophozoite, and segmenter stages; major transcription occurs in the late trophozoite/early schizont stage. This is consistent with the high glutathione reductase activity found in early schizonts. Other data also suggest that PfGR2 corresponds to the enzyme isolated from parasitized erythrocytes. These criteria include the subunit molecular mass (56.2 kDa), the N-terminal sequence (VYDLIVIGGGSGGMA), the presence of specific sequence motifs at ligand-binding sites, and, as demonstrated by Western blotting, the occurrence of a unique chain segment in the core of the central domain. In view of these data, the function(s) of PfGR2 as well as PfGR1, the product of another GR-like gene of P. falciparum (Müller et al., 1995) should be carefully assessed.

Molecular and biochemical characterization of a Plasmodium falciparum cyclophilin containing a cleavable signal sequence.

The immunosuppressive drug cyclosporin A (CsA) inhibits the growth of malaria parasites in vitro and in vivo. Cyclosporin A exerts its immunosuppressive effect in T lymphocytes by binding to cyclophilin (CyP), a peptidylprolyl cis-trans isomerase (PPIase). It is believed that the cyclosporin/cyclophilin complex inhibits a Ca(2+)-activated protein phosphatase, calcineurin, involved in T-cell activation. A cDNA encoding a cyclophilin of the human malaria parasite Plasmodium falciparum has been isolated as a step in the elucidation of the mechanism of antimalarial action of CsA. This cDNA, termed PfCyP, encodes a protein of 195 amino acids which has highest similarity with the Candida albicans (73.1%) and the Drosophila melanogaster (73.1%) cytoplasmic cyclophilins. A Northern blot reveals an approximately 900-bp nucleotide transcript that is consistent with the predicted size of the encoded polypeptide. The predicted PfCyP protein has a putative endoplasmic-reticulum-directed signal sequence at its N-terminus and two potential N-linked glycosylation sites. Expression of PfCyP RNA in an in vitro translation/translocation system reveals that the PfCyP protein is translocated across microsomes, that the signal peptide is cleaved and that the PfCyP protein is glycosylated at two sites. The PfCyP cDNA open reading frame coding for the predicted mature protein has been expressed in Escherichia coli. The purified recombinant protein is an active PPIase (kcat/Km = 2.3 x 10(6) s-1 M-1); this enzymic activity is inhibited by CsA (IC50 = 10 nM). The PfCyP protein has thus the same sensitivity to CsA as the PPIase activity associated with P. falciparum extracts [Bell, A. et al. (1994) Biochem. Pharmacol. 48, 495-503] suggesting that PfCyP may be responsible for the PPIase activity in those extracts. If different cyclophilins exist in P. falciparum, we conclude that either the PfCyP protein is the major cyclophilin detected in the parasite or that there are other cyclophilins with similar susceptibilities to CsA.

A Plasmodium falciparum protein kinase with two unusually large kinase inserts.

A protein kinase gene (PfPK1) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The gene, which contains one intron, encodes a protein with a predicted length of 909 amino acids. The predicted protein contains all the conserved sequences characteristic of a protein kinase catalytic domain. These sequences are discontinuous, however, since they are separated by two large kinase inserts with 178 and 330 amino acids in size. Specific antisera were raised against recombinant fragments of the protein and a PfPK1-specific peptide. Using one of these antibodies, a functional protein kinase was precipitated from malarial lysates and this kinase recognized casein as an exogenous substrate. PfPK1 was expressed in a stage-specific fashion and also had a stage-specific cellular localization. During the intraerythrocytic life cycle, PfPK1 shifts from the parasite cytosol to the parasite membrane fraction. An unusual feature of PfPK1 is its electrophoretic mobility on SDS-PAGE. Whereas the predicted protein size is about 100 kDa, the apparent size is about 70 kDa. There are no indications for RNA processing and we could exclude proteolytic processing as an explanation.

Expression and secretion of malarial parasite beta-tubulin in Bacillus brevis.

Microtubule inhibitors are active against the human malarial parasite Plasmodium falciparum, but whether these drugs actually interact with parasite tubulins is not known. It has not previously been possible to produce mg quantities of isolated, soluble tubulin subunits for drug-binding experiments. A cDNA encoding P falciparum beta-tubulin was expressed and the protein secreted in Bacillus brevis. With the addition of EGTA to the culture medium, which increases shedding of proteins from the cell surface, up to 2 mg/l recombinant beta-tubulin was obtained in supernatants. It is not clear why B brevis is able to secrete this normally cytoplasmic protein, but the secretion levels of recombinant proteins may be related to the net charge of the first few residues of the mature polypeptide.

Isolation of a novel Plasmodium falciparum gene encoding a protein homologous to the Tat-binding protein family.

We have cloned a Plasmodium falciparum gene that belongs to the nuclear Tat-binding protein (TBP) gene family. This gene, PfTBP, is (A + T)-rich and encodes a 49.5-kDa protein. The predicted protein encoded by this gene has highest similarity to the slime mold protein DdTBP10 (86%) and to the yeast protein SUG1 (81.8%), both of which belong to the Tat-binding protein family. In agreement with the characteristics of this family, PfTBP contains a highly conserved domain of approximately 200 amino acids, in which are found the motifs A and B of ATPases, and amino acid sequences characteristic of a large family of RNA or DNA helicases, suggesting a role in RNA or DNA unwinding. Like DdTBP10, the PfTBP protein has a heptad repeat of four leucine residues, reminiscent of a leucine zipper motif known to mediate dimerization. We have further characterized PfTBP gene expression by Northern-blot analysis. This gene is expressed in a stage-specific manner, with higher expression in the late trophozoite stage. The recombinant PfTBP gene has been expressed in Escherichia coli and a polyclonal antiserum has been raised in rabbits against the recombinant protein. This antibody has been used to study the protein in the parasite. The gene product is expressed in a stage-specific manner with higher expression in the late trophozoite and schizont stages, and is localized in the nucleus of the erythrocytic stage parasite. Thus the protein might have a function in DNA synthesis and/or in transcription, as is the case for other Tat-binding proteins.

Plasmodium falciparum calcium-dependent protein kinase phosphorylates proteins of the host erythrocytic membrane.

The unusual Ca(2+)-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62,000, is activated by Ca2+ (K0.5 = 15 microM) in the presence of Mg2+ or Mn2+, and can associate with 45Ca2+. The activation by Ca2+ could be partially replaced by Mn2+, but not by Zn2+ or Mg2+. PfCPK preferentially phosphorylated casein and histone H1. The Km and Vmax for Mg2+ ATP were 26 microM and 70 nmol min-1 mg-1, respectively, with casein as substrate; and 34 microM and 143 nmol min-1 mg-1, respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.

Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin.

The immunosuppressive peptide cyclosporin A inhibits the growth of malaria parasites in vitro and in vivo, but little is known about its mechanism of antimalarial action. The immunosuppressive action of cyclosporin A is believed to result from binding of the drug to cyclophilins (intracellular peptidyl-prolyl cis-trans isomerases), and inhibition of the protein phosphatase calcineurin by the cyclosporin A-cyclophilin complex. Two immunosuppressive macrolides, FK506 and rapamycin, bind to a distinct isomerase, FKBP12, and the FK506-FKBP complex also inhibits calcineurin. Calcineurin itself is apparently involved in signal transduction between the T-cell membrane and nucleus, and its inhibition blocks T-cell activation. Rapamycin inhibits a later step in T-cell proliferation. Peptidyl-propyl cis-trans isomerase activity was detected in extracts of Plasmodium falciparum. It was completely inhibited by concentrations of cyclosporin A above 0.1 microM, but not by FK506 or rapamycin, and probably represented one or more cyclophilins. Comparison of the antimalarial and anti-isomerase activities of a series of cyclosporin analogues failed to reveal a correlation between the two properties. Cyclosporin A and its more active 8'-oxymethyl-dihydro-derivative, in combination with the cyclophilin-containing P. falciparum extract, inhibited the protein phosphatase activity of bovine calcineurin. Therefore inhibition of a putative P. falciparum calcineurin by a complex of CsA and cyclophilin might be responsible for the antimalarial action of the drug. The most active cyclosporin, however, was a 3'-keto-derivative of cyclosporin D (SDZ PSC-833) which inhibited P. falciparum growth with a 50% inhibitory concentration (IC50) of 0.032 microM (compared with 0.30 microM for cyclosporin A), but was a poor inhibitor of the parasite isomerase. 3'-Keto-cyclosporin D has negligible immunosuppressive activity, but it strongly inhibits the P-glycoprotein of multi-drug resistant mammalian tumour cells. FK506 and rapamycin were also active antimalarials (IC50 of 1.9 and 2.6 microM, respectively) but in the absence of detectable FKBP in P. falciparum extracts, their mechanisms of antimalarial action remain unclear.

Calcium-binding properties of a calcium-dependent protein kinase from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation.

Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.

Two major phosphoproteins of Plasmodium falciparum are heat shock proteins.

Two major phosphoproteins of Plasmodium falciparum could be identified by partial amino acid sequencing as the plasmodial members of the hsp 70 heat shock protein family, Pfhsp and Pfgrp. According to phosphoamino acid analyses of Pfhsp and Pfgrp isolated from [32P]orthophosphate-labeled malarial cultures, both proteins were phosphorylated in Ser and Thr. While Pfhsp contains higher amounts of labeled phosphoserine, Pfgrp contains higher amounts of phosphothreonine. Phosphorylation of both proteins increased throughout the entire erythrocytic growth cycle. At the trophozoite and schizont stages Pfhsp and Pfgrp are the most prominent phosphoproteins of Plasmodium falciparum. Using multiply redundant oligonucleotides directed against the N-terminus of Pfgrp we cloned and sequenced the entire Pfgrp gene. The gene encodes a product with a predicted length of 652 amino acids. The deduced amino acid sequence has identities of 65.5% and 65.0% to the human and rat grp78 proteins, respectively. Pfgrp possesses a classical N-terminal leader sequence. The published grp78 related gene sequences of Plasmodium falciparum are all fragments of the same plasmodial gene.

Visual disability resulting from a dislocated intraocular lens.

Complications may arise from the failure to remove a dislocated intraocular lens, even when it appears to rest in a benign position and pose no problems. We examined a patient with a second intraocular lens left in the inferior vitreous cavity. Shifting the loose lens resulted in eventual complications and visual disability; removing the dislocated lens and replacing the functional intraocular lens yielded good results.

Lacrimal gland-derived lymphocyte proliferation potentiating factor.

PURPOSE: To determine whether lacrimal gland acinar cells are involved in modulating local immune response; to isolate and characterize a lacrimal gland factor that exerts biological activities on T lymphocytes. METHODS: A protein factor has been purified from lacrimal gland extracts by a combination of ion exchange and gel-filtration chromatography. This factor has the capacity to enhance proliferation of T lymphocytes upon stimulation with a mitogen or an antigen. We have, therefore, called this substance lacrimal gland-derived lymphocyte proliferation potentiating factor (LG-F). RESULTS: Lacrimal gland-derived lymphocyte proliferation potentiating factor has a molecular weight of approximately 65,000 daltons as determined by gel-filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. T cells demonstrate greater proliferation when cultured with high concentrations of concanavalin A (Con A) in the presence of LG-F, as compared with culture without addition of LG-F. This enhancing effect of LG-F may be mediated by IL-2, because the final cell count correlates with the levels of IL-2 secreted by LG-F-activated cells. Lacrimal gland-derived lymphocyte proliferation potentiating factor is nonmitogenic for T cells, but its potentiating effect is antigen-dependent. Dual stimulation of OA-primed T cells with both OA and LG-F results in greater proliferative activity, in contrast to culture with OA alone. CONCLUSIONS: The results suggest that lacrimal gland cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production. The presence of such a factor in the lacrimal gland may prove to be of importance in the generation of local immune responses.

Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins.

An unusual protein kinase gene, termed PfCPK, was isolated from Plasmodium falciparum. The gene, which contains five exons and four introns, encodes a product with a predicted length of 524 amino acids. The amino-terminal segment of the predicted protein contains all of the conserved sequences characteristic of a protein kinase catalytic domain and has a high homology to several protein serine-threonine kinase subfamilies (30-41% amino acid identities). These subfamilies include calcium/calmodulin-dependent protein kinases, calcium-dependent protein kinase, ribosomal S6 protein kinase, cyclic nucleotide-dependent protein kinases, protein kinase C, and the yeast SNF1 subfamily. All of these protein kinases are relatively close in the phylogeny tree and within the kinase catalytic domains have about 35% amino acid identities to each other, suggesting that PfCPK is also in this region of the phylogeny tree. An unusual feature of PfCPK is that its carboxyl-terminal segment displays homology to the EF hand calcium-binding proteins, for example 34% amino acid identity to chicken fast skeletal muscle troponin C and 35% amino acid identity to human calmodulin. Like troponin Cs and calmodulins, PfCPK also contains four EF hand calcium-binding motifs. Furthermore, the four introns in the PfCPK gene are all located in the carboxyl-terminal putative EF hand calcium-binding region (EF hand calcium-binding proteins from higher eukaryotes generally contain multiple introns). This combination of a protein kinase and an EF hand calcium-binding protein in a single polypeptide implies that PfCPK may be directly activated by calcium. Constructs containing the full-length PfCPK cDNA have been expressed in Escherichia coli at a high level to generate a 60-kDa recombinant protein. Compared with similar fractions from control cells, the fraction containing PfCPK recombinant protein exhibited an elevated protein kinase activity which was Ca(2+)-dependent.

Effects of microtubule inhibitors on protein synthesis in Plasmodium falciparum.

At low concentrations, both isomers of tubulozole (C, T) inhibit Plasmodium falciparum but only tubulozole C inhibits mammalian cells. Since tubulozole C prevents polymerization of mammalian tubulin whereas tubulozole T does not, the antimalarial action of tubulozoles may not involve microtubules. The present study concerns the inhibition of parasite protein synthesis by the tubulozoles. While tubulozoles took 3-4 h to kill parasites in erythrocytic culture, they inhibited protein synthesis within 10 min. The concentrations of the drug required were, however, too high for this to account for their antimalarial action. The microtubule inhibitor colcemid inhibited protein synthesis rapidly and at relevant concentrations, but vinblastine did not inhibit protein synthesis. Tubulozole T and colcemid inhibited protein synthesis posttranscriptionally since they had little effect on RNA synthesis. Analysis of labelled parasite proteins by two-dimensional gel electrophoresis showed that while it inhibited synthesis of most proteins to the same degree, tubulozole T super-inhibited the synthesis of certain proteins. This may cause its antimalarial effect at low concentrations.