A Miocene to Pleistocene climate and elevation record of the Sierra Nevada (California).

Orographic precipitation of Pacific-sourced moisture creates a rain shadow across the central part of the Sierra Nevada (California) that contrasts with the southern part of the range, where seasonal monsoonal precipitation sourced to the south obscures this rain shadow effect. Orographic rainout systematically lowers the hydrogen isotope composition of precipitation (deltaD(ppt)) and therefore deltaD(ppt) reflects a measure of the magnitude of the rain shadow. Hydrogen isotope compositions of volcanic glass (deltaD(glass)) hydrated at the earth's surface provide a unique opportunity to track the elevation and precipitation history of the Sierra Nevada and adjacent Basin and Range Province. Analysis of 67 well dated volcanic glass samples from widespread volcanic ash-fall deposits located from the Pacific coast to the Basin and Range Province demonstrates that between 0.6 and 12.1 Ma the hydrogen isotope compositions of meteoric water displayed a large (>40 per thousand) decrease from the windward to the leeward side of the central Sierra Nevada, consistent with the existence of a rain shadow of modern magnitude over that time. Evidence for a Miocene-to-recent rain shadow of constant magnitude and systematic changes in the longitudinal climate and precipitation patterns strongly suggest that the modern first-order topographic elements of the Sierra Nevada characterized the landscape over at least the last 12 million years.

CpABC, a Cryptosporidium parvum ATP-binding cassette protein at the host-parasite boundary in intracellular stages.

The intracellular parasite Cryptosporidium parvum develops inside a vacuole at the apex of its epithelial host cell. The developing parasite is separated from the host cell cytoplasm by a zone of attachment that consists of an extensively folded membranous structure known as the feeder organelle. It has been proposed that the feeder organelle is the site of regulation of transport of nutrients and drugs into the parasite. In this report, we localize an approximately 200-kDa integral membrane protein, CpABC, from Cryptosporidium parvum to the host-parasite boundary, possibly the feeder organelle. The predicted amino acid sequence of CpABC has significant structural similarity with the cystic fibrosis conductance regulator and the multidrug resistance protein subfamily of ATP-binding cassette proteins. This is an example of a parasite-encoded transport protein localized at the parasite-host interface of an intracellular protozoan.

Cyclosporin analogs inhibit in vitro growth of Cryptosporidium parvum.

Cyclosporine and nonimmunosuppressive cyclosporin (CS) analogs were demonstrated to be potent inhibitors of the growth of the intracellular parasite Cryptosporidium parvum in short-term (48-h) in vitro cultures. Fifty-percent inhibitory concentrations (IC50s) were 0.4 microM for SDZ 033-243, 1.0 microM for SDZ PSC-833, and 1.5 microM for cyclosporine. Two other analogs were less effective than cyclosporine: the IC50 of SDZ 205-549 was 5 microM, and that of SDZ 209-313 was 7 microM. These were much lower than the IC50 of 85 microM of paromomycin, a standard positive control for in vitro drug assays for this parasite. In addition, intracellular growth of excysted sporozoites that had been incubated for 1 h in cyclosporine was significantly reduced, suggesting that the drug can inhibit sporozoite invasion. The cellular activities of the CS analogs used have been characterized for mammalian cells and protozoa. The two analogs that were most active in inhibiting C. parvum, SDZ PSC-833 and SDZ 033-243, bind weakly to cyclophilin, a peptidyl proline isomerase which is the primary target of cyclosporine and CS analogs. However, they are potent modifiers of the activity of the P glycoproteins/ multidrug resistance (MDR) transporters, members of the ATP-binding cassette (ABC) superfamily. Hence, both cyclophilin and some ABC transporters may be targets for this class of drugs, although drugs that preferentially interact with the latter are more potent. Cyclosporine (0.5 microM) had no significant chemosensitizing activity. That is, it did not significantly increase sensitivity to paromomycin, suggesting that an ABC transporter is not critical in the efflux of this drug. Cyclosporine at concentrations up to 50 microM was not toxic to host Caco-2 cells in the CellTiter 96 assay. The results of this study complement those of studies of the inhibitory effect of cyclosporine and CS analogs on other apicomplexan parasites, Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii.

Preferential binding of Plasmodium falciparum SERA and rhoptry proteins to erythrocyte membrane inner leaflet phospholipids.

Proteins of an apical organelle, the rhoptry, of Plasmodium falciparum are secreted into the host erythrocyte membrane during merozoite invasion. To identify the membrane-binding site for rhoptry proteins, we examined the binding of parasite proteins to phospholipid vesicles. A specific interaction between the rhoptry proteins of 140, 130, and 110 kDa to vesicles containing phosphatidylserine and phosphatidylinositol was observed. Both phospholipids are preferentially localized on the inner leaflet of the bilayer. Binding to other phospholipids, including sphingomyelin, was considerably less. In addition, the 120-kDa serine repeat antigen known as SERA, which was determined to be present on the merozoite, bound to phosphatidylserine vesicles and much less to vesicles of other phospholipids. Both the rhoptry and SERA proteins exhibited a preference for phosphatidylserine with short acyl side chains. Specific binding of SERA and the rhoptry proteins to phospholipids of the inner leaflet of membranes suggests a possible mechanism by which the protein facilitate invasion into host cells.

Biogenesis of rhoptry organelles in Plasmodium falciparum.

Biogenesis of the rhoptry organelle of Plasmodium falciparum was studied by examining the synthesis and assembly of rhoptry proteins at different stages of intraerythrocytic development. Rhoptry proteins examined in this study were those of the high molecular weight complex of 140/130/110 kDa and referred to as Rhop-H1,2,3 and the low molecular weight complex of 80 and 42 kDa referred to as Rhop-L1,2. Co-ordinate, stage-specific expression of three proteins, Rhop-H3, Rhop-L1 and Rhop-L2, was observed; maximum levels of mRNA at the 8 nucleus stage correlated with the onset of protein synthesis. In contrast, mRNA levels for DNA polymerase-alpha, a marker for DNA replication during schizogony, was maximum just prior to the onset of the first nuclear division, indicating that rhoptry biogenesis is not co-ordinate with nuclear division. The assembly of newly synthesized rhoptry proteins was followed by subcellular fractionation of schizonts at different stages of development. At the four-nucleus stage a vesicle could be isolated by sucrose gradient fractionation which had a peak density of 1.12 g ml-1 and contained only Rhop-H2 and Rhop-H3 proteins. This vesicle could represent an intermediate or pre-rhoptry compartment. At the 8-nucleus stage, the Rhop-L1 protein was also detected in a vesicle of low density. At the 16-nucleus stage, the proteins were present in vesicles having a significantly greater density in sucrose, 1.16 g ml-1, similar to that of the mature organelle. The study suggested that the rhoptry proteins first accumulate in a low density vesicle and that assembly into this compartment is staggered. Immunoelectronmicroscopy studies indicated that the Rhop-H3 protein is first present in small granular compartments that becomes more electron dense and enlarges due to the stage-dependent incorporation of proteins.

Transfer of a dense granule protein of Plasmodium falciparum to the membrane of ring stages and isolation of dense granules.

A 14-kDa protein was localized to the dense granules of Plasmodium falciparum by immunoelectron microscopy with monoclonal antibody 1H1. The protein was present in dense granules in late-stage schizonts and free merozoites. After invasion, the protein was localized exclusively on the membrane of the newly invaded ring. The protein is referred to as RIMA, for ring membrane antigen. The 14-kDa protein was synthesized late in schizogony as determined by immunofluorescence microscopy and immunoblotting. At the late schizont stage it was distributed diffusely throughout the intracellular schizont. Only at the segmenter stage was the protein localized in defined spots that correspond to dense granules. Dense granules were isolated from schizont-infected erythrocytes by subcellular fractionation on a sucrose gradient. Fractions containing the 14-kDa protein were detected by immunoblotting with monoclonal antibody 1H1. The 14-kDa protein was first detected in vesicles at the late (8-nucleus) schizont stage. Mature dense granules sedimented with a peak density of 1.17 g/ml, which is similar to the density of rhoptries isolated by the same procedure.

Rhoptry organelles of apicomplexan parasites.

Rhoptries are the unifying structural feature of the intracellular, opicomplexon parasites and are implicated in having a central role in host cell invasion. Ultrastructural studies of zoites of different genera suggest that the participation of rhoptries in the invasion of the respective host cells is morphologically similar. However, biochemical analysis of their protein constituents reveals a considerable degree of diversity between different coccidion parasites. In this article Margaret Perkins asks whether there are common structural determinants of the rhoptry components of different genera and if the underlying mechanism of rhoptry function is similar in all opicomplexon parasites.

Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes.

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.

Isolation and characterization of rhoptries of Plasmodium falciparum.

Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml-1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an Mr of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 x g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na2CO3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.

The kinetic depth effect and optic flow--II. First- and second-order motion.

We use a difficult shape identification task to analyze how humans extract 3D surface structure from dynamic 2D stimuli--the kinetic depth effect (KDE). Stimuli composed of luminous tokens moving on a less luminous background yield accurate 3D shape identification regardless of the particular token used (either dots, lines, or disks). These displays stimulate both the 1st-order (Fourier-energy) motion detectors and 2nd-order (nonFourier) motion detectors. To determine which system supports KDE, we employ stimulus manipulations that weaken or distort 1st-order motion energy (e.g. frame-to-frame alternation of the contrast polarity of tokens) and manipulations that create microbalanced stimuli which have no useful 1st-order motion energy. All manipulations that impair 1st-order motion energy correspondingly impair 3D shape identification. In certain cases, 2nd-order motion could support limited KDE, but it was not robust and was of low spatial resolution. We conclude that 1st-order motion detectors are the primary input to the kinetic depth system. To determine minimal conditions for KDE, we use a two frame display. Under optimal conditions, KDE supports shape identification performance at 63-94% of full-rotation displays (where baseline is 5%). Increasing the amount of 3D rotation portrayed or introducing a blank inter-stimulus interval impairs performance. Together, our results confirm that the human KDE computation of surface shape uses a global optic flow computed primarily by 1st-order motion detectors with minor 2nd-order inputs. Accurate 3D shape identification requires only two views and therefore does not require knowledge of acceleration.

Nonadditivity of masking by narrow-band noises.

Characterization of the visual system as a linear system has many consequences. One property implied by such a characterization is that signal-to-noise ratio at threshold is constant, so that the contrast energy of a sinusoidal grating at threshold depends on the effective noise passed by the filter used to detect the target. When the contrast energy in an external noise is sufficiently high, the contribution of internal noise may be conveniently ignored. In such circumstances, the effectiveness of a given noise is measured by its ability to mask the target. One consequence of the linearity assumption is that if the energy of the effective noise is increased by a factor k, then threshold energy of the signal is increased by the same amount. The contrast energy at threshold in the presence of a masker created by adding two maskers should be the sum of the threshold energies in the individual maskers. We have tested this hypothesis for spectrally nonoverlapping maskers. We find that the contrast energy required to detect the target in the presence of the combined maskers is much greater than the sum of the threshold energies for the two maskers. This "excess masking" violates the linearity assumption. On the other hand, when maskers do have spectral overlap, no excess masking is found.

Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion.

Rhoptry proteins of Plasmodium falciparum merozoites, of 140, 130, and 110 kDa, identified by co-precipitation with Mab.1B9, bind selectively to mouse erythrocytes and reticulocytes. The properties of binding are shown to correlate with invasion of P. falciparum into mouse erythrocytes. Invasion of two strains of P. falciparum 7G8 and FCR-3, into mouse erythrocytes was examined, and was found to differ significantly. The 7G8 strain invades mouse erythrocytes at a rate of 40-60% compared to invasion into human erythrocytes, whereas FCR-3 invades at a rate of 5-15%. Both strains of P. falciparum preferentially invade reticulocytes in the in vitro invasion assay. This correlated with an increase in the amount of rhoptry protein of the 7G8 strain bound to mouse erythrocytes, compared to the FCR-3 strain and an increased binding to reticulocytes compared to mature erythrocytes. Binding of the rhoptry proteins and merozoite invasion into the erythrocyte is blocked in erythrocytes treated with trypsin and chymotrypsin but not in neuraminidase-treated erythrocytes, suggesting that the putative receptor site is exposed and accessible on the erythrocyte surface. Rabbit antiserum against gp3, the major glycophorin of mouse erythrocytes, blocks binding of the rhoptry proteins to erythrocytes and reduces merozoite invasion into mouse erythrocytes by 50%. Binding of rhoptry proteins to mouse reticulocytes was not blocked by alpha gp3 indicating a receptor difference between reticulocytes and erythrocytes. Mab.1B9 reduces merozoite invasion but does not decrease binding of the rhoptry proteins to the mouse erythrocyte. The mouse erythrocyte serves as a useful model to study the receptor-ligand interaction of rhoptry proteins and host surface proteins and to define the role of the rhoptry proteins during the invasion process.

Chemical crosslinking of Plasmodium falciparum glycoprotein, Pf200 (190-205 kDa), to the S-antigen at the merozoite surface.

Merozoites were isolated from Plasmodium falciparum cultures labeled with [3H]mannose and [35S]methionine and treated with a cleavable homobifunctional crosslinker, dithiobis(succinimidyl) propionate. The crosslinked complexes were immunoprecipitated with Mab.5B1 directed against the major merozoite surface glycoprotein. Pf200 (MW 190-205), and reduced with dithiothreitol. Crosslinked immunocomplexes did not contain the second major merozoite surface glycoprotein, Pf50 (MW 45-55 kDa), or other major [35S]methionine-labeled proteins, except for a weakly labeled protein of 150 kDa. Crosslinked complexes immunoprecipitated with Mab.5B1 and then reduced with DTT were immunoblotted with antibody directed against three soluble P. falciparum antigens, a serine-rich antigen known as Pf126 or SERA, the S-antigen, and GBP-130. The 150-kDa S-antigen was readily detected in crosslinked immunocomplexes with Pf200. The SERA antigen, although crosslinked under these conditions, was not detected in association with Pf200 nor was GBP-130.

Phosphorylation of erythrocyte membrane and cytoskeleton proteins in cells infected with Plasmodium falciparum.

Phosphorylation changes in the erythrocyte membrane and cytoskeletal proteins as a consequence of infection by the malarial parasite Plasmodium falciparum were examined. Spectrin, band 3, band 4.1, ankyrin and glycophorin are phosphorylated in normal erythrocytes. As a consequence of invasion by the merozoite, the extracellular stage of the parasite, into 32P-prelabeled normal erythrocytes, all the major 32P-labeled erythrocyte proteins are dephosphorylated. As the parasite develops intracellularly from the immature ring stage to the mature schizont stage, selective phosphorylation of certain host proteins, spectrin, ankyrin and band 3 is observed. Band 4.1 does not appear to incorporate [32P]phosphate at any stage of parasite development. These observed phosphorylation changes may be important in the regulation of the cytoskeletal organization in P. falciparum-infected cells.

Localization of a parasite encoded protein to erythrocyte cytoplasmic vesicles of Plasmodium falciparum-infected cells.

Intracellular development of the malarial parasite results in substantial modifications of the membrane and cytoskeleton of the erythrocyte host cell. Two related Plasmodium falciparum-encoded proteins of 50 kDa and 43 kDa (Pf 50/43), identified by reactivity with a single mAb, were demonstrated to be localized to the erythrocyte cytoplasm of parasite-infected cells. Immunofluorescence and immunoelectron microscopy using mAb.7E11 demonstrated the Pf 50/43 is localized in the membrane of the vesicles in the erythrocyte cytoplasm, vesicles which correspond to Maurer's clefts. Solubility properties of the proteins suggest they are integral membrane proteins. By immunofluorescence, Pf 50/43 is shown to colocalize with actin which has a highly modified organization in the infected erythrocyte. Pf 50/43 is located exclusively in the vesicles, is not transported to the erythrocyte membrane or secreted. It is proposed the vesicles may play a role in transport of molecules across the erythrocyte cytoplasm, between the parasite and the external erythrocyte plasma membrane.

Erythrocyte invasion by two Plasmodium falciparum isolates differing in sialic acid dependency in the presence of glycophorin A antibodies.

Merozoites of Plasmodium falciparum depend on glycophorins for invasion into human erythrocytes, although this dependency varies between different geographic isolates of the species. The FCR-3 (Gambia) isolate appears to be fully dependent on the N-acetylneuraminic acid (NeuNAc) residues of the O-linked tetrasaccharide of glycophorin for invasion. Invasion of the CDC-1 (Honduras) isolate into neuraminidase treated erythrocytes is 50% of that into normal erythrocytes. This and additional results suggest that this isolate is not fully dependent on the O-linked saccharides of glycophorin. In the present study, invasion of CDC-1 and FCR-3 isolates into erythrocytes was examined in the presence of Fab fragments of monoclonal antibodies directed against different domains of glycophorin. Fab fragments directed against the carbohydrate domain inhibited invasion of both isolates but inhibited invasion by the FCR-3 isolate more than CDC-1 isolate. The reactivity of monoclonal antibodies (Mabs) directed against the carbohydrate domain was dependent on the NeuNAc residues as binding was abolished in neuraminidase treated erythrocytes. Mabs directed against the peptide domain of glycophorin A did not significantly inhibit invasion by either isolate. These results are consistent with other findings that the CDC-1 isolate is not fully dependent on the carbohydrate domain of glycophorin for invasion.