Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3

Ca²⁺ signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca²⁺ stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [γ-³²P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone II_AS as a representative substrate in a Ca²⁺-dependent manner at a high Ca²⁺ concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.     

Gametogenesis in Malaria Parasites Is Mediated by the cGMP-Dependent Protein Kinase

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca²⁺) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.     

Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca²⁺) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.     

Biosorption of acid violet dye from aqueous solutions using native biomass of a new isolate of Penicillium sp.

Laboratory investigation of the potential use of Penicillium sp. as biosorbent for the removal of acid violet dye from aqueous solution was studied with respect to pH, temperature, biosorbent, initial dye concentrations. Penicillium sp. decolourizes acid violet (30 mg l⁻¹) within 12 h agitation of 150 rpm at pH 5.7 and temperature of 35 °C. The pellets exhibited a high dye adsorption capacity (5.88 mg g⁻¹) for acid violet dye over a pH range (4–9); the maximum adsorption was obtained at pH 5.7. The increase of temperature favored biosorption for acid violet, but the optimum temperature was 35 °C. Adsorption kinetic data were tested using pseudo-first-order, pseudo-second-order and kinetic studies showed that the biosorption process follows pseudo-first-order rate kinetics with an average rate constant of 0.312 min⁻¹. Isotherm experiments were conducted to determine the sorbent–desorption behavior of examined dye from aqueous solutions using Langmuir and Freundlich equations. Langmuir parameter indicated a maximum adsorption capacity of 4.32 mg g⁻¹ for acid violet and R_L value of 0.377. Linear plot of log q_e vs log C_e shows that applicability of Freundlich adsorption isotherm model. These results suggest that this fungus can be used in biotreatment process as biosorbent for acid dyes.     

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca²⁺ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (K_m = 4.45 ± 1.46 mM) than for the diphenol, l-DOPA (K_m = 10.27 ± 1.33 mM). Maximum enzyme activity was evident at 37 °C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.     

Enzymatic characterization of the Plasmodium vivax chitinase, a potential malaria transmission-blocking target

The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (β-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc₃ compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at ~ 25 °C) activity profiles and sensitivity to allosamidin (IC₅₀ = 6 µM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1.     

2-Hydroxyacid dehydrogenase from Haloferax mediterranei, a D-isomer-specific member of the 2-hydroxyacid dehydrogenase family

An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei. The enzyme is a dimer with a molecular mass of 101.4 ± 3.3 kDa. It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl. The enzyme is characterized by a broad substrate specificity 2-ketoisocaproate and 2-ketobutyrate being the substrates with the higher V_max/K_m. When pyruvate and 2-ketobutyrate were the substrates the optimal pH was acidic (pH 5) meanwhile for 2-ketoisocaproate maximum activity was achieved at basic pH between 7.5 and 8.5. The optimum temperature was 52 ºC and at 65 ºC there was a pronounced activity decrease. This new enzyme can be used for the production of D-2-hydroxycarboxylic acid.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Diet and physiological responses of Spondyliosoma cantharus (Linnaeus, 1758) to the Caulerpa racemosa var. cylindracea invasion

Marine invasions are a worldwide problem that involves changes in communities and the acclimation of organisms to them. The invasive Chlorophyte Caulerpa racemosa var. cylindracea is widespread in the Mediterranean and colonises large areas from 0 to 70 m in depth. The omnivorous fish Spondyliosoma cantharus presents a high frequency of occurrence of C. racemosa in the stomach contents at invaded areas (76.3%) while no presence of C. racemosa was detected in control areas. The isotopic composition of muscle differed significantly between invaded and non-invaded sites for δ¹³C (− 16.67‰ ± 0.09 and − 17.67‰ ± 0.08, respectively), δ¹⁵N (10.22‰ ± 0.22 and 9.32‰ ± 0.18, respectively) and the C:N ratio (2.01 ± 0.0002 and 1.96 ± 0.009, respectively). Despite the high frequency of occurrence of C. racemosa in the stomach contents of S. cantharus and its important contribution to the δ¹³C source (20.7% ± 16.2), the contribution of C. racemosa to the δ¹⁵N in S. cantharus food sources was very low (6.6% ± 5.8). Other invertebrate prey such as decapods and polychaetes were more important contributors to the δ¹⁵N source at both invaded and non-invaded sites. Activation of enzymatic pathways (catalase, superoxide dismutase, glutathione-s-tranferase, 7-ethoxy resorufin O-de-ethylase) but not a significant increase in lipid peroxidation MDA (0.49 ± 0.01 nmol/mg prot at non-invaded and 0.53 ± 0.01 nmol/mg prot at invaded sites) was observed in S. cantharus individuals living in C. racemosa-invaded sites compared with control specimens. The low δ¹⁵N contribution values of C. racemosa by S. cantharus together with the toxicity demonstrated by the activation of the antioxidant defences and the important contribution of invertebrate prey to the δ¹⁵N could mean that the ingestion of C. racemosa by S. cantharus might be unintentional during the predation of invertebrate preys living underneath the entanglement of the C. racemosa fronds and stolons mats.     

Purification and characterisation of the cardenolide-specificβ-glucohydrolase CGH II from Digitalis lanata leaves

A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg^–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The K_m- and V_max-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg^–1 protein, respectively.     

Growth and protein synthesis of barramundi, Lates calcarifer, fed lupin as a partial protein replacement

Protein synthesis is an essential growth process in all animals. Little information is available on post-prandial protein synthesis and even less where different protein sources are compared. Protein synthesis was measured at 4 and 24 h after feeding juvenile barramundi in order to determine the effect of using lupin as a partial protein replacement for fish meal on the post-prandial protein metabolism. Juvenile barramundi (4.3 ±0.6 g) were held in a recirculation system (27 °C, salinity 10‰ and 24 h light) for 15 days. Fish were fed one of two isonitrogenous isoenergetic diets (40% crude protein, 16% lipid and 18.5 GE MJ kg^− 1). One diet was formulated with 100% fish meal as the protein source while the other had 45% of the protein replaced with lupin ingredients (lupin kernel meal (Lupinus angustifolius) and lupin protein concentrate). All fish were fed a ration of 6%·d^− 1 and feed intake was not significantly different between the two diets. Specific growth rate (SGR) and growth efficiency (in relation to protein (PPV) and energy (PEV)) were 6.5 ± 0.14%·d^− 1, 43.8 ± 2.72% and 38.31 ± 1.56%, respectively, and were not significantly different between the two diets. There was no significant difference in protein synthesis between the two diets at 4 and 24 h after feeding, however protein synthesis was significantly higher 4 h after feeding than at 24 h (p = 0.02). Neither growth performance nor protein metabolism was altered by replacing 45% of the protein with lupin protein and indicated this to be a suitable protein source for barramundi feeds.     

Genetic Distance in Housekeeping Genes Between <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> and <Emphasis Type="Italic">Plasmodium reichenowi</Emphasis> and Within <Emphasis Type="Italic">P. falciparum</Emphasis>

The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672±0.0088 and 0.0011±0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30±5.40) × 10⁴ and (11.62±7.56) × 10⁴ years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively.This article contains an online supplementary table.Reviewing Editor: Dr. Martin Kreitman     

Dynamic changes in white blood cell counts in uncomplicated Plasmodium falciparum and P. vivax malaria

Total and differential white blood cell (WBC) counts are basic and essential indicators in any type of illness resulting from infection. In malaria, WBC counts are generally characterized as low to normal during treatment. WBC-counts data, before and during treatment with artemisinin derivatives, was gathered for patients with either Plasmodium falciparum or Plasmodium vivax infection (at 28-day follow-up), to investigate dynamic changes in WBC count. We analyzed and compared the WBC counts of 1310 inpatients presenting with uncomplicated P. falciparum and P. vivax malaria at the Hospital for Tropical Diseases, in Bangkok, Thailand. Before-treatment, a statistically significant negative correlation was found between initial WBC count and highest temperature on admission. Before and during treatment, WBC counts were significantly lower in P. falciparum than P. vivax infection on days 0 and 7, but the numerical difference was small. We also found clinically significantly low WBC counts during the acute stages of both types of malaria, which subsequently normalized by day 28 follow-up. This finding has important clinical implications for the conventional method of estimating parasitemia using an assumed WBC count of 8000 cells/μL. The most significant finding in our analysis is that WBC counts in acute P. falciparum and P. vivax malaria are significantly lower than previously assumed for estimating malaria-parasite density. However, these abnormalities returned to normal within several weeks after artemisinin-derivative-based treatment.     

Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR

The effect of refrigerated and frozen storage on the viability of Vibrio vulnificus was evaluated using cell suspensions (1 × 10⁸ CFU/ml). Ethidium bromide monoazide (EMA) was utilized to selectively allow real-time (Rti) PCR amplification of target DNA from viable but not dead cells. Bacterial survivors from the EMA Rti-PCR were evaluated by comparison with the plate count assay following different temperature exposures (− 20 and 4 °C) every 24 h for 72 h. The log CFU values from the EMA Rti-PCR assays were erroneously higher than that from plate counts. DNA amplification was not completely suppressed by EMA treatment of low temperature destroyed cells suggesting that membrane damage was not sufficient to allow effective EMA penetration into the cells. The optimal concentration of sodium deoxycholate (SD) was also determined to enhance discrimination of viable and dead cells following exposure of cells to low temperatures. The use of 0.01% or less of SD did not inhibit the Rti-PCR amplification derived from viable bacterial cells. A rapid decrease of the log CFU was observed with cell suspensions subjected to frozen storage and a slow decline in the log CFU occurred at 4 °C. The combination of SD and EMA treatments applied to cells of V. vulnificus held at − 20 °C and 4 °C resulted in a high level of correlation between the log of CFU (plate counts) and the log of the number of viable cells determined from SD+EMA Rti-PCR.     

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 °C to 0 °C, 8 °C, 16 °C, 24 °C, and 32 °C for 72 h) and salinity changes (from 30‰ to 20‰, 25‰, and 35‰ for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25‰ and 35‰) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 °C. At 32 °C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 °C for 0.5 h exposure, and increased significantly at 32 °C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 °C in 0.5 h to 6 h exposures and at 8 °C for 1 h. By contrast, activities of MPO decreased significantly in 24 °C and 32 °C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 °C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20‰. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses.     

Pfs 16 pivotal role in Plasmodium falciparum gametocytogenesis: A potential antiplasmodial drug target

Mature gametocytes, the sexual stage of Plasmodium falciparum, ensure the continued transmission of malaria from the human host to the mosquito vector. Even if gametocytes are not implicated in the malaria physiopathology it is crucial to the spread of malaria. Gametocytes are to be a key target for drugs used against Plasmodium in public health. The expression levels of 4 sexual-stage specific genes, Pfs 16, Pfs 25, Pfg 27and S 18S rRNA, during gametocytogenesis of various P. falciparum strains were analyzed by a real time PCR assay. The strains showed different capacities to produce mature gametocytes and in parallel different patterns of sexual gene expression. There was a correlation only between Pfs 16 cDNA overexpression in the first 48 h of the culture and the production of mature gametocytes. Pfs 16 is an early marker of the development of mature gametocytes in cultures and is therefore a potential target for new antimalarial drugs.     

Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus)

The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O₂⁻ into the stabler H₂O₂. In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 °C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 °C and completely heat inactivated at 80 °C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.     

Thermotolerance and gene expression following heat stress in the whitefly Bemisia tabaci B and Q biotypes

The whitefly Bemisia tabaci (Gennadius) causes tremendous losses to agriculture by direct feeding on plants and by vectoring several families of plant viruses. The B. tabaci species complex comprises over 10 genetic groups (biotypes) that are well defined by DNA markers and biological characteristics. B and Q are amongst the most dominant and damaging biotypes, differing considerably in fecundity, host range, insecticide resistance, virus vectoriality, and the symbiotic bacteria they harbor. We used a spotted B. tabaci cDNA microarray to compare the expression patterns of 6000 ESTs of B and Q biotypes under standard 25 °C regime and heat stress at 40 °C. Overall, the number of genes affected by increasing temperature in the two biotypes was similar. Gene expression under 25 °C normal rearing temperature showed clear differences between the two biotypes: B exhibited higher expression of mitochondrial genes, and lower cytoskeleton, heat-shock and stress-related genes, compared to Q. Exposing B biotype whiteflies to heat stress was accompanied by rapid alteration of gene expression. For the first time, the results here present differences in gene expression between very closely related and sympatric B. tabaci biotypes, and suggest that these clear-cut differences are due to better adaptation of one biotype over another and might eventually lead to changes in the local and global distribution of both biotypes.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.     

Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca²⁺ (1.25 up to 11.25 mM) to force generation at 25 °C (acclimation temperature), 15 and 35 °C. The post-rest tension and force–frequency relationship were conducted with and without 10 μM ryanodine in the bathing medium. Increments in extracellular Ca²⁺ resulted in increases in twitch force development only at 35 °C. A significant post-rest potentiation was recorded for the control preparations at 25 °C (100% to 119.8 ± 4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 °C (100% to 97.6 ± 1.5%). At 35 °C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6 ± 4.6%) while at 15 °C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 °C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.