Disassociation of ß1-α1-ß2 from the α2-α3 domain of prion protein (PrP) is a prerequisite for the conformational conversion of PrPC into PrPSc: Driven by the free energy landscape.

Misfolding of the cellular prion protein (PrPC) into ß-sheet-rich scrapie form (PrPSc) is associated with transmissible spongiform encephalopathies. A point mutation F198S is responsible for the development of a rare inherited Gerstmann-Straussler-Scheinker disease caused by the aggregation of PrPC. Thus, identification and the structural characterization of aggregation-prone regions are essential to delineate the conversion of PrPC to the disease-associated PrPSc upon F198S mutation. In the present study, molecular dynamics simulations on the wild-type PrP (WT-PrP) and its mutant were performed to explore the structural basis responsible for aggregation driven by the mutation. Secondary structure analysis revealed that the mutant exhibited a partial unfolding on α2 and the complete distortion in the 310-helix of the ß2-α2 loop. Remarkably, the ß2-α2 loop is in proximity to α3 attributed by the long-range hydrophobic interactions and such structural intimacy is not observed in the WT-PrP. Owing to this, the ß1-α1-ß2 regions have separated from α2-α3 domain resulting in the impairment on the hydrogen bond between α1 and α3. Thus, the present study provides a detailed structural description of the F198S mutant in line with previous experimental results and delivers insights into the structural basis responsible for the conversion of PrPC to the disease-associated PrPSc.

Glaucoma Pred: Glaucoma prediction based on Myocilin genotype and phenotype information.

Glaucoma is the second leading cause of blindness after cataract and is heterogeneous in nature. Employing a genetic approach for the detection of the diseased condition provides an advantage that the gene responsible for the disease can be identified by genetic test. The availability of predictive tests based on the published literature would provide a mechanism for early detection and treatment. The genotype and phenotype information could be a valuable source for predicting the risk of the disease. To this end, a web server has been developed, based on the genotype and phenotype of myocilin mutation, which were identified by familial linkage analysis and case studies. The proposed web server provides clinical data and severity index for a given mutation. The server has several useful options to help clinicians and researchers to identify individuals at a risk of developing the disease. Glaucoma Pred server is available at http://bioserver1.physics.iisc.ac.in/myocilin.

Polymorphisms in an intronic region of the myocilin gene associated with primary open-angle glaucoma--a possible role for alternate splicing.

PURPOSE: To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. METHODS: Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. RESULTS: A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the MYOC gene showed the presence of the predicted g.14072G>A polymorphism, g.1293C/T heterozygous polymorphism, instead of our predicted g.1293C/- polymorphism. Other than the prediction, two novel SNPs (g.1295G>T and g.1299T>G) and two reported SNPs (g.1284G>T and g.1286G>T) were also identified. Cluster analysis showed the g.14072G>A homozygous condition was more common in this cohort than the heterozygous condition. CONCLUSIONS: We previously proposed that the disruption of dimer or oligomer formation by the C-term region allows greater chances of nucleation for aggregation. Here we suggest that polymorphisms in the myocilin genomic region that cause synonymous codon changes or those that occur in the intron regions can possibly lead to altered myocilin protein products through altered intron-exon splicing.

Parasite plastids: maintenance and functions.

Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.

SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima.

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.

Antibiotic inhibitors of organellar protein synthesis in Plasmodium falciparum.

Elongation factor Tu (EF-Tu) is encoded by the tuf gene of the plastid organelle of the malaria parasite Plasmodium falciparum. A range of structurally unrelated inhibitors of this GTP-dependent translation factor was shown to have antimalarial activity in blood cultures. The most active was the cyclic thiazolyl peptide amythiamicin A with an IC50 = 0.01 microM. Demonstrable complexes were formed in vitro between a recombinant version of P. falciparum EF-Tu(pl) and inhibitors that bind to different sites on EF-Tu; these included the antibiotics kirromycin, GE2270A and enacyloxin IIa.

Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum.

Malaria parasites, and other parasitic protists of the Phylum Apicomplexa, carry a plastid-like genome with greatly reduced sequence complexity. This 35 kb DNA circle resembles the plastid DNA of non-photosynthetic plants, encoding almost exclusively components involved in gene expression. The complete gene map described here includes genes for duplicated large and small subunit rRNAs, 25 species of tRNA, three subunits of a eubacterial RNA polymerase, 17 ribosomal proteins, and a translation elongation factor. In addition, it codes for an unusual member of the Clp family of chaperones, as well as an open reading frame of unknown function found in red algal plastids. Transcription is polycistronic. This plastid-like DNA molecule is conserved in several genera of apicomplexans and is conjectured to have been acquired by an early progenitor of the Phylum by secondary endosymbiosis. The function of the organelle (plastid) carrying this DNA remains obscure, but appears to be specified by genes transferred to the nucleus.

Phylogenetic analysis of the rpoB gene from the plastid-like DNA of Plasmodium falciparum.

Malaria and other Apicomplexan parasites harbour two extrachromosomal DNAs. One is mitochondrial and the other is a 35-kb circle with some plastid-like features but whose provenance and function is unknown. In addition to genes for rRNAs, tRNAs and ribosomal proteins, the 35-kb circular DNA of Plasmodium falciparum carries an rpoBC operon which encodes subunits of a eubacteria-like RNA polymerase. The phylogenetic analysis of the complete rpoB sequence presented here supports our inference that the 35-kb circle is the remnant of a plastid genome.

Nine duplicated tRNA genes on the plastid-like DNA of the malaria parasite Plasmodium falciparum.

A major feature of the plastid-like circular DNA of Plasmodium falciparum is an inverted repeat comprising duplicated genes for rRNA (rrn) and tRNA (trn). We have identified nine putative trn genes in each arm of the repeat on the basis of their potential clover-leaf structures and conserved residues. Northern blots indicate that these trn genes are expressed.

The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum: new evidence supports a possible rhodophyte ancestry.

In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.

Sequence and organization of large subunit rRNA genes from the extrachromosomal 35 kb circular DNA of the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum carries an extrachromosomal 35 kb circular DNA molecule of unknown provenance. A striking feature of the circle is a palindromic sequence of genes for subunit rRNAs and several tRNAs, spanning ca. 10.5 kb. The palindrome has an intriguing resemblance to the inverted repeat of plastid genomes, and the sequence and putative secondary structure of the malarial large subunit (LSU) rRNA described in this report were used as the basis of a phylogenetic study. The malarial rRNA was found to be highly divergent in comparison with a selected group of chloroplast LSU rRNAs but was more closely related to them than to mitochondrial LSU rRNA genes.

Effect of intra-erythrocytic magnesium ions on invasion by Plasmodium falciparum.

Exclusion of magnesium ions from resealed ghosts or their extraction from intact human red cells by means of an ionophore results in a reversible drop in susceptibility to invasion by Plasmodium falciparum merozoites in vitro. Resealed ghosts, containing magnesium-ATP and diluted cytosol, are invaded with high efficiency only when the original hypotonic lysis is carried out in the presence of magnesium ions. This effect is not related to the loss of membrane-associated constituents when magnesium ions are absent. Ghosts containing calcium ions, together with the protective agent, flunarizine, were essentially resistant to invasion; this effect is again at least partially reversible. A possible explanation of these phenomena is that entry of the merozoite may be inhibited by breakdown of the host cell phospholipid asymmetry, with the appearance of aminophospholipids at the outer cell surface.

A study of red cell membrane properties in relation to malarial invasion.

The shape and mechanical properties of human red cells were modified in several ways and the consequences for the efficiency of invasion by Plasmodium falciparum in culture were investigated. Inhibition of invasion by depletion of ATP was shown to be unrelated to cell shape or deformability changes. Treatment of cells with N-ethylmaleimide (NEM), which dissociates some 70% of the native spectrin tetramers into the dimer, grossly reduced deformation of the cells under shear and increased by a factor of two or more the shear elastic modulus, as measured by the micropipette aspiration technique. Cells thus treated were efficiently invaded by P. falciparum (ca. 75% of control). In a population of cells pretreated with chlorpromazine, parasites were found in stomatocytic cells which were highly undeformable under shear. There was also considerable invasion into cells from subjects with hereditary pyropoikilocytosis, and two types of elliptocytosis. Cells treated with wheat germ agglutinin showed a dose-dependent increase in rigidity; a fivefold increase in elastic modulus (with total loss of deformation under shear in our conditions) still permitted invasion at a level of 50% of the control. The results suggest that gross mechanical properties of the membrane per se, at least within any physiologically relevant range, are unlikely to be the primary determinant of malarial invasion; this may instead be linked to the freedom of membrane proteins to migrate in the course of entry of the parasite.

Transcutaneous monitors during one-lung ventilation: are they reliable?

During one-lung ventilation, levels of oxygen and carbon dioxide in the blood are commonly assessed by intermittent blood gas sampling. Transcutaneous PO2 (tcPO2) and transcutaneous PCO2 (tcPCO2) have been reported to accurately reflect arterial PO2 (PaO2) and arterial PCO2 (PaCO2) in hemodynamically stable patients. Transcutaneous monitors appear to be ideal for detecting trends toward hypoxia and hypercarbia, conditions that may not be evident when using intermittent blood gas sampling, while pulse oximetry, since it reflects saturation, may not detect hypoxia until it has already occurred. Thirty: one patients undergoing one-lung ventilation were monitored using both transcutaneous electrodes applied to the upper arm (group 1) or chest (group 2) and arterial blood gas sampling. Arterial blood gases were sampled while tcPO2 and tcPCO2 values were being recorded. Regression, correlation, and covariance analyses were performed. Correlation coefficients of PaO2 to tcPO2 varied from .05 to .99 for each patient. The slopes of individual regression lines varied from 0.03 to 1.16. Correlation coefficients of PaCO2 to tcPCO2 varied from .01 to .99, while the slopes of individual regression lines ranged from 0.02 to 5.89. Covariance analyses revealed considerable variation in PaO2 to tcPO2 and PaCO2 to tcPCO2 in individual patients even under stable hemodynamic conditions. Analysis of covariance also demonstrated that in group 2 the slopes comparing arterial and transcutaneous values were significantly different for PaO2 <100 mmHg and PaO2 >200 mmHg. In group 1, for PaO2 <100 mmHg, there was no difference in slopes but y-intercepts were significantly different (P < .05). However, transcutaneous indices were significantly different in both groups for PaO2 <100 mmHg and PaO2 >200 mmHg. It is concluded that transcutaneous monitoring is useful to indicate trends in arterial values in some patients, but blood gas analysis is still necessary to verify the reliability of such monitoring.

Cytoplasmic factor required for entry of malaria parasites into RBCs.

Resealed ghosts of human RBCs, containing diluted cytosol, are susceptible to invasion by Plasmodium falciparum. If ATP is present, a dilution of up to about 30-fold, corresponding to an intracellular hemoglobin concentration of approximately 10 mg/mL, can be tolerated without total loss of susceptibility to invasion. Up to a dilution of about one-half this, the parasites also develop normally. When the cytosol is diluted by more than the critical amount, invasion of the resulting resealed ghosts falls off abruptly. If the diluent buffer is replaced by extraneous concentrated hemolysate, an indefinite dilution is possible without loss of invasion. There is thus an intracellular constituent, which must be present at a concentration above some critical level if the parasite is to enter the cell. The factor in question is not dialyzable. It is largely inactivated when the hemolysate is kept for approximately 1 day in the cold or for approximately 20 minutes at 45 degrees C. The inability of a heat-treated hemolysate to support invasion is not due to the generation of inhibitory products, because such a solution can be used as a diluent of a fresh hemolysate without inhibition of invasion. When the inactivated hemolysate is present as a major component, however, the parasites fail to develop to the trophozoite stage. The invasion-linked factor remains in the strongly adsorbed nonheme fraction when a batchwise separation from hemoglobin on an anion exchanger is made and is thus probably acidic in character; the adsorbed fraction, recovered from the ion-exchanger, substantially restores capacity for invasion when sealed into ghosts. Its activity is destroyed by treatment with trypsin. The adsorbed fraction contains many proteins. When fractionated on a gel filtration column by fast liquid chromatography, active material eluted at a volume corresponding to a mol wt for a globular protein in the region of 10,000. A component of apparent subunit mol wt of 13,000 was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this eluate fraction.

Inhibition of malarial invasion by intracellular antibodies against intrinsic membrane proteins in the red cell.

It has previously been shown that antibodies against the transmembrane proteins, band 3 and glycophorin A, inhibit entry of the merozoite into the red cell and, in the case of band 3, it was established that attachment of the parasite to the cell is not prevented. We have found that antibodies against the cytoplasmic domains of band 3 and of glycophorin A, when present in the interior of resealed ghosts of human red cells, also inhibit invasion by P. falciparum. It is inferred that attachment of the merozoite to the red cell causes structural effects that are transduced to the membrane cytoskeleton and the antibodies against transmembrane proteins interfere with the invasion sequence at this level.

Plasmodium falciparum: protease inhibitors and inhibition of erythrocyte invasion.

Invasion of human red blood cells by Plasmodium falciparum is inhibited by the protease inhibitors, leupeptin and chymostatin. The efficacy of chymostatin was reduced if the cells were first treated with chymotrypsin. On the other hand, exposure of fresh cells to the supernatant from a synchronous culture at the reinvasion stage showed no such effect. This suggests that a proteolytic step occurs in the course of invasion and may be confined to the region of contact between the invading parasite and the erythrocyte. To test this, leupeptin or chymostatin was introduced into lysed cells, which were then resealed. The intracellular inhibitor strongly reduced invasion. Leupeptin also caused a striking effect on the development of the trophozoite stage of the parasites: a massive vacuole, apparently containing undigested haemoglobin, developed within the parasite. This did not totally stop development and the vacuolated parasites could be recovered in relatively pure form by lysis of the parasitised host cells with saponin.

Control of malarial invasion by phosphorylation of the host cell membrane cytoskeleton.

It has been shown that the entry of the malaria parasite into the red blood cell requires the presence of ATP in the host cell cytoplasm. In red blood cell ghosts that contain no ATP the receptor on the extracellular surface remains in place and parasites will bind to the membrane, but will not enter. ATP is thus necessary for one of the steps in the invasion sequence that follows recognition and attachment. The process of entry appears to involve the active participation of the host cell membrane cytoskeleton. We have suggested that the function of the intracellular ATP may be to regulate phosphorylation of the cytoskeleton. We now present evidence that the activity of the membrane-associated cyclic AMP-independent kinase of the red blood cell is inseparable from invasion; the active substrate may be spectrin.