The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium.

During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5'-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.

(Table 2) Stratigraphic marker taxa in ODP Hole 162-985A sediments

The rich and diverse dinocyst assemblages in Cores 162-985A-32X through 62X confirm the importance of these microfossils in unraveling the evolution of the Norwegian Sea. Cosmopolitan taxa, with well-documented stratigraphic ranges in northwest Europe, indicate the following ages: Sections 162-985A-62X-1 through 51X-2, Rupelian (early Oligocene); 50X-5, Oligocene, possibly Chattian; 48X-6, Aquitanian? (early Miocene); 48X-4 through 37X-5, Aquitanian (early Miocene); and 36X-5 through 32X-1, Burdigalian (early Miocene). This stratigraphic interpretation suggests that a major hiatus, which can be correlated with an apparently coeval hiatus at Site 643, occurs within the Chattian at Site 985. Several endemic dinocyst taxa with unusual morphology and restricted stratigraphic occurrences are present in Hole 985A and other Norwegian Sea sites, especially Site 643. By using Hole 985A data for control, the Oligocene-Miocene sediments can be correlated with some degree of confidence in the Norwegian Basin.

(Table T1) Correlations between marker beds and drill holes for ODP Leg 171B sites

More than 50 discrete volcanic ash layers were recovered at the five drill sites of the Blake Nose depth transect (Leg 171B, western central Atlantic). The majority of these ash layers are intercalated with Eocene hemipelagic sediments with a pronounced frequency maximum in the upper Eocene. Several ash layers appear to be deposited from volcanic fallout with little or no indication of secondary remobilization. They provide excellent stratigraphic markers for a correlation of the Leg 171B drill sites. Other ash layers were probably redeposited from volcaniclastic-rich turbidity currents, but they still represent geologically instantaneous events that can be used in stratigraphic correlation between adjacent drill holes. Additional nonvolcanic marker beds, like the suspect late Eocene impact event layer, were included in our hole-to-hole correlations. Stratigraphic and downcore positions of marker beds were compiled and plotted against existing composite depth records that were constructed to guide high-resolution sampling. Comparison of our correlation with the spliced composite sections of each drill site reveals several minor and some major discrepancies. These may result from drilling distortion or missing sections, from the lack of unambiguous criteria for the synchronism of ash layers, or from the systematic exclusion of marker-bed data in the construction of the spliced record. Integration of both correlation approaches will help eliminate most of the observed discrepancies.

Host-parasite relationships of the clover cyst nematode, Heterodera trifolii Goffart /

Cover title.

Statistical evaluation of the effectiveness of Federal Motor Vehicle Safety Standard 108: Side Marker Lamps (Only). Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Final design and implementation plan for evaluating the effectiveness of FMVSS 108: side marker lamps and high intensity headlamps (only). Tasks #4 and #5 report.

National Highway Traffic Safety Administration, Washington, D.C.