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Biomedical subjects

Jeffrey R Johnson

Publications and source records attributed to Jeffrey R Johnson.

10 recordsLinked to original sources

A systematic map of genetic variation in Plasmodium falciparum.

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified approximately 500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs.

ATP-Binding Cassette Transporters↗

Optimal timing of ampicillin administration to pregnant women for establishing bactericidal levels in the prophylaxis of Group B Streptococcus.

OBJECTIVE: The purpose of this study was to determine the time necessary to achieve and maintain bactericidal concentrations of ampicillin in the cord blood. STUDY DESIGN: This was a prospective study in which women scheduled for an elective cesarean section were given intravenous ampicillin before the procedure. Cord and simultaneous maternal blood samples were collected at the time of delivery. Serum ampicillin levels were analyzed by high-performance liquid chromatography. RESULTS: The ratio of cord blood to maternal serum ampicillin concentration was found to increase over time in a linear fashion. There was no correlation between maternal body mass index (22.3 to 48.3 kg/m2) and ampicillin cord blood concentrations. All cord serum samples far exceeded minimal bactericidal concentrations reported for Group B Streptococcus (0.25 to 2.0 microg/mL), even at 338 minutes following administration. CONCLUSION: Bactericidal levels of ampicillin in the cord blood are rapidly achieved within 30 minutes of administration of ampicillin to the mother. The increase in the ratio of cord to maternal serum ampicillin levels is directly related to time, suggesting a decrease in the clearance of ampicillin in the newborns as compared to the mothers. The cord blood ampicillin concentration exceeds the maternal concentration and both continue to be above the minimal bactericidal concentrations at 5.6 hours after administration. No relationship was observed between the maternal body mass index and ratio of cord to maternal serum concentrations of ampicillin.

Adult↗

An integrated view of the chemistry and mineralogy of martian soils.

The mineralogical and elemental compositions of the martian soil are indicators of chemical and physical weathering processes. Using data from the Mars Exploration Rovers, we show that bright dust deposits on opposite sides of the planet are part of a global unit and not dominated by the composition of local rocks. Dark soil deposits at both sites have similar basaltic mineralogies, and could reflect either a global component or the general similarity in the compositions of the rocks from which they were derived. Increased levels of bromine are consistent with mobilization of soluble salts by thin films of liquid water, but the presence of olivine in analysed soil samples indicates that the extent of aqueous alteration of soils has been limited. Nickel abundances are enhanced at the immediate surface and indicate that the upper few millimetres of soil could contain up to one per cent meteoritic material.

Bromine↗

A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling.

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.

Amino Acid Sequence↗

Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle.

To investigate the role of post-transcriptional controls in the regulation of protein expression for the malaria parasite, Plasmodium falciparum, we have compared mRNA transcript and protein abundance levels for seven different stages of the parasite life cycle. A moderately high positive relationship between mRNA and protein abundance was observed for these stages; the most common discrepancy was a delay between mRNA and protein accumulation. Potentially post-transcriptionally regulated genes are identified, and families of functionally related genes were observed to share similar patterns of mRNA and protein accumulation.

Animals↗

The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion.

The first gene characterizing the clag (cytoadherence linked asexual gene) family of Plasmodium falciparum was identified on chromosome 9. The protein product (Clag9) was implicated in cytoadhesion, the binding of infected erythrocytes to host endothelial cells, but little information on the biochemical characteristics of this protein is available. Other genes related to clag9 have been identified on different chromosomes. These genes encode similar amino acid sequences, but clag9 shows least conservation. Clag9 was detected in schizonts, merozoites and ring-stage parasites after protease digestion and peptide analysis by mass spectrometry. Using antisera raised against unique regions of Clag9 and against RhopH2, a component of the RhopH high-molecular-mass protein complex of merozoites, immunofluorescence co-localized the two proteins to the apical region of merozoites. Immunoelectron microscopy co-localized Clag9 and RhopH2 exclusively to the basal bulb region of rhoptries rather than to their apical ducts. The same Clag9-specific antibodies bound the RhopH complex, and the protein was detected in the complex purified by antibodies to RhopH2. Clag9 protein was also shown to be present in ring-stage parasites, carried through from the previous cycle with the RhopH complex, in a location identical to that of RhopH2. Transcription of the clag9 gene was shown to occur at the same time as the genes for other members of the RhopH complex, rhoph2 and 3. The results indicate that Clag9 is part of the RhopH complex and suggest that, within this complex, the protein previously designated RhopH1 is composed of more than one protein product of the clag gene family. The results cast doubt on a direct role for Clag9 in cytoadhesion; we suggest that the primary role of the RhopH complex is in remodelling the infected red blood cell after invasion by the merozoite. The complex may have multiple functions dependent on its exact composition, which may include, with respect to Clag9, a contribution to the mechanism of cytoadhesion.

Amino Acid Sequence↗

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry.

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.

Algorithms↗

Fourier-transform mass spectrometry for automated fragmentation and identification of 5-20 kDa proteins in mixtures.

When presented with a mixture of intact proteins, electrospray ionization with Fourier-transform mass spectrometry (ESI-FTMS) has the capability to obtain direct fragmentation information from isolated ions. However, the automation of this capability has not been achieved to date. We have developed software for unattended acquisition of protein tandem mass spectrometry (MS/MS) data and batch processing of the resulting files for identification of whole proteins. Mixtures of both protein standards (8-29 kDa) and Methanococcus jannaschii cytosolic proteins (up to six components + 20 kDa) were infused via an autosampler, and MS/MS data were acquired without human intervention. The acquisition software recognizes ESI charge state patterns, generates protein-specific isolation waveforms on-the-fly, and fragments ions using two different infrared laser times. In addition to protein standards, five wild-type proteins (7-14 kDa) were identified automatically with 100% sequence coverage from the M. jannaschii database. The software underpins a measurement platform for sample-dependent acquisition of MS/MS data for whole proteins, a critical step to realize proteomics with 100% sequence coverage in a higher throughput setting.

Archaeal Proteins↗

Proteomics in malaria.

The recent completion of human, Anopheles gambiae, and Plasmodium falciparum genomes relevant to the study of human malaria allows the application of modern proteomic technologies to complement previously implemented conventional approaches. Proteomic analysis has been employed to elucidate global protein expression profiles, subcellular localization of gene products, and host-pathogen interactions that are central to disease pathogenesis and treatment. The high-throughput nature of these techniques is in accord with the pace of drug and vaccine development that have the potential to directly reduce the morbidity and mortality of disease.

Animals↗

Mean arterial pressure in extremely low birth weight concordant and discordant twins during the first day of life.

OBJECTIVE: To determine mean arterial pressure (MAP) values during the first 24 hours for stable concordant and discordant extremely low birth weight (ELBW) twins and to ascertain its association with perinatal factors. BACKGROUND: In ELBW infants, whether singletons or concordant or discordant twins, hypotension is diagnosed by nonspecific clinical signs together with reference arterial pressure values extrapolated from regression models or from scarce actual observations. DESIGN: Retrospective cohort study. METHODS: We studied 26 sets of concordant and 29 sets of discordant twins, one of whom in each set weighed < or = 800 g at birth. Infants with umbilical cord hemoglobin > or = 14 g/dl and who, although mechanically ventilated, had normal acid-base balance, no patent ductus arteriosus, had not received indomethacin, steroids, muscle relaxants, narcotics, were never treated for hypotension, and survived at least 7 days were considered stable. Arterial pressures were determined by oscillometry (OBP) and direct transducer readings using an umbilical line (MAP). All admission and 10 % of the subsequent readings were measured by OBP; the remaining were measured by MAP. RESULTS: Concordant and discordant twins were similar in demographics, history of chorioamnionitis, preeclampsia, antepartum steroids, cesarean delivery, and neonatal morbidity, but were different in mean birth weight (700 and 789 g), and gestational age (GA) (25 and 27 weeks). Forty-four (82%) of all concordant and 14 (26%) of 58 discordant twins were treated empirically for hypotension. Head ultrasounds were normal or showed Grade I/II in 74% concordant, 81% discordant, and 80% discordant infants with twin-to-twin transfusion syndrome (TTTX). Neonatal mortality was 46%, 45%, and 47%, respectively. There were 14 stable concordant and 22 stable discordant. Their MAPs were different at 1 hour (29 and 34 Torr), 3 hours (29 and 35 Torr), 6 hours (30 and 37 Torr), 12 hours (31 and 36 Torr), 18 hours (33 and 35 Torr), and 24 hours (34 and 36 Torr), respectively. Twenty-six small and 26 large concordant infants had similar MAP from the 1st (27 and 28 Torr) to the 24th hour of life (43 and 43 Torr). Concordant males (often not stable) had lower MAP than concordant females. Seventeen small discordant twins had lower MAP from 1 to 24 hours (28 and 33 Torr) than 17 large discordant twins without TTTX (32 and 38 Torr). Small discordant twins with (donors) and without TTTX had similar trends and MAP values. Large discordant twins with TTTX (recipient) had the highest MAP from birth to 24 hours than any other subgroup of infants and, unlike the others, the MAP trend decreased over time. MAP correlated with GA but not with very low birth weight (< or = 750 g), although with the same GA, those with higher birth weights had higher MAP, and at the same birth weight younger GA twins had lower MAP values. CONCLUSION: MAP increases from birth to 24 hours in all concordant and discordant twins regardless of condition (stable or unstable), birth weight (large or small) or GA. Recipient TTTX twins had higher MAP throughout but, unlike the other twins, it declined over 24 hours. Small discordant and donor TTTX infants should be considered intrauterine growth restricted and are expected to have MAP commensurable to their GA and not to their birth weight.

Apgar Score↗