Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Adherence to Chemoprophylaxis and Plasmodium falciparum Anti-Circumsporozoite Seroconversion in a Prospective Cohort Study of Dutch Short-Term Travelers

Background

We conducted a prospective study in a cohort of short-term travelers assessing the incidence rate of anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad.

Methods

Adults were recruited from the travel clinic of the Public Health Service Amsterdam. They kept a structured daily travel diary and donated blood samples before and after travel. Blood samples were serologically tested for the presence of Plasmodium falciparum anti-circumsporozoite antibodies.

Results

Overall, the incidence rate (IR) of anti-circumsporozoite seroconversion was 0.8 per 100 person-months. Of 945 travelers, 620 (66%) visited high-endemic areas and were advised about both chemoprophylaxis and preventive measures against mosquito bites. Most subjects (520/620 = 84%) took at least 75% of recommended prophylaxis during travel. Travel to Africa, use of mefloquine, travel duration of 14–29 days in endemic areas, and concurrent use of DEET (N,N-diethyl-meta-toluamide) were associated with good adherence practices. Four travelers without fever seroconverted, becoming anti-circumsporozoite antibody-positive. All four had been adherent to chemoprophylaxis; two visited Africa, one Suriname, one India. Ten subjects with fever were tested for malaria while abroad and of these, three received treatment. All three were adherent to chemoprophylaxis and tested negative for anti-circumsporozoite antibodies.

Conclusion

Travel to Africa, using mefloquine, travel duration of 14–29 days in endemic areas, and use of DEET were associated with good adherence to chemoprophylaxis. The combination of chemoprophylaxis and other preventive measures were sufficient to protect seroconverting travelers from clinical malaria. Travelers who were treated for malaria abroad did not seroconvert.     

Recombinant human antibodies specific for the Pfs48/45 protein of the malaria parasite Plasmodium falciparum

We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one V(H) family-derived germ-line gene (V(H)1) and two V(L) family segments (V(L)2 and V(K)I).     

The dynamics of naturally acquired immune responses to Plasmodium falciparum sexual stage antigens Pfs230 & Pfs48/45 in a low endemic area in Tanzania

Background

Naturally acquired immune responses against sexual stages of P. falciparum can reduce the transmission of malaria from humans to mosquitoes. These antigens are candidate transmission-blocking vaccines but little is known about the acquisition of sexual stage immunity after exposure to gametocytes, or their longevity and functionality. We conducted a longitudinal study on functional sexual stage immune responses.

Methodology/Principal Findings

Parasitaemic individuals (n = 116) were recruited at a health centre in Lower Moshi, Tanzania. Patients presented with gametocytes (n = 16), developed circulating gametocytes by day 7 (n = 69) or between day 7 and 14 (n = 10) after treatment or did not develop gametocytes (n = 21). Serum samples were collected on the first day of gametocytaemia and 28 and 84 days post-enrolment (or d7, 28, 84 after enrolment from gametocyte-negative individuals). Antibody responses to sexual stage antigens Pfs230 and Pfs48/45 were detected in 20.7% (72/348) and 15.2% (53/348) of the samples, respectively, and were less prevalent than antibodies against asexual stage antigens MSP-1₁₉ (48.1%; 137/285) and AMA-1 (52.4%; 129/246)(p<0.001). The prevalence of anti-Pfs230 (p = 0.026) and anti-Pfs48/45 antibodies (p = 0.017) increased with longer duration of gametocyte exposure and had an estimated half-life of approximately 3 months. Membrane feeding experiments demonstrated a strong association between the prevalence and concentration of Pfs230 and Pfs48/45 antibodies and transmission reducing activity (TRA, p<0.01).

Conclusions/Significance

In a longitudinal study, anti-Pfs230 and Pf48/45 antibodies developed rapidly after exposure to gametocytes and were strongly associated with transmission-reducing activity. Our data indicate that the extent of antigen exposure is important in eliciting functional transmission-reducing immune responses.     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc^15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients.     

Nucleotide variation in the triosephosphate isomerase (Tpi) locus of Drosophila melanogaster and Drosophila simulans

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.      

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.