Prospects for the Development of Odour Baits to Control the Tsetse Flies Glossina tachinoides and G. palpalis s.l.

Field studies were done of the responses of Glossina palpalis palpalis in Côte d''Ivoire, and G. p. gambiensis and G. tachinoides in Burkina Faso, to odours from humans, cattle and pigs. Responses were measured either by baiting (1.) biconical traps or (2.) electrocuting black targets with natural host odours. The catch of G. tachinoides from traps was significantly enhanced (∼5×) by odour from cattle but not humans. In contrast, catches from electric targets showed inconsistent results. For G. p. gambiensis both human and cattle odour increased (>2×) the trap catch significantly but not the catch from electric targets. For G. p. palpalis, odours from pigs and humans increased (∼5×) the numbers of tsetse attracted to the vicinity of the odour source but had little effect on landing or trap-entry. For G. tachinoides a blend of POCA (P = 3-n-propylphenol; O = 1-octen-3-ol; C = 4-methylphenol; A = acetone) alone or synthetic cattle odour (acetone, 1-octen-3-ol, 4-methylphenol and 3-n-propylphenol with carbon dioxide) consistently caught more tsetse than natural cattle odour. For G. p. gambiensis, POCA consistently increased catches from both traps and targets. For G. p. palpalis, doses of carbon dioxide similar to those produced by a host resulted in similar increases in attraction. Baiting traps with super-normal (∼500 mg/h) doses of acetone also consistently produced significant but slight (∼1.6×) increases in catches of male flies. The results suggest that odour-baited traps and insecticide-treated targets could assist the AU-Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) in its current efforts to monitor and control Palpalis group tsetse in West Africa. For all three species, only ∼50% of the flies attracted to the vicinity of the trap were actually caught by it, suggesting that better traps might be developed by an analysis of the visual responses and identification of any semiochemicals involved in short-range interaction.     

Effect of osmolarity on LDL binding and internalization in hepatocytes

The present study has been performed to elucidate a possiblerole of cell volume in low-density lipoprotein (LDL) binding andinternalization (LDL_b+i). Asshown previously, increase of extracellular osmolarity (OSMe) andK⁺ depletion, both known to shrinkcells, interfere with the formation of clathrin-coated pits and thuswith LDL_b+i. On the other hand,alterations of cell volume have been shown to modify lysosomal pH,which is a determinant of LDL_b+i.LDL_b+i have been estimated fromheparin-releasable (binding) or heparin-insensitive (internalization)uptake of ¹²⁵I-labeled LDL. OSMewas modified by alterations of extracellular concentrations of ions,glucose, urea, or raffinose. When OSMe was altered by varying NaClconcentrations, LDL_b+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe andLDL_b+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to alteredaffinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 µg/ml at 219, 293, and 435 mosM, respectively. A 25%increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreasedlysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMedecreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by0.17 ± 0.02) pH. Partial replacement of extracellularNa⁺ withK⁺ had little effect onLDL_b+i, although it swelledhepatocytes and increased lysosomal and cytosolic pH. Hypertonicglucose, urea, or raffinose did not exert similar effects despite ashrinking effect of hypertonic raffinose. Monensin, which completelydissipates lysosomal acidity, virtually abolishedLDL_b+i. In conclusion, theobservations reveal a significant effect of ionic strength onLDL_b+i. The effect is, however,not likely to be mediated by alterations of cell volume or alterationsof lysosomal pH.

     

Circadian rhythm of cardiac bradyarrhythmia episodes in rats

It was investigated whether incidence of S-A block and A-V block (type I and II) observed in male Wistar rats shows a circadian periodicity. Under the 14/10 light-dark illumination schedule, circadian periodicity was demonstrated in the occurrence of bradyarrhythmias as well as slow wave sleep, paradoxical sleep locomotive activity and heart beats/hour. This circadian variation of bradyarrhythmia shows two major peaks. The highest peak coincides with the time zone immediately after the start of illumination, and the second peak coincides with the acrophase of the circadian variation in paradoxical sleep. The 25-h period of circadian rhythm of bradyarrhythmias was disclosed even under constant illumination. This circadian variation shows the disappearance of the early peak of two peaks observed in the light-dark schedule. These results show the existence of an endogenous circadian rhythm in bradyarrhythmias as well as in sleep-wakefulness and locomotive activity. This knowledge about the circadian rhythm of bradyarrhythmia must be incorporated into the effective treatment of arrhythmias.     

Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.     

How Do Tsetse Recognise Their Hosts? The Role of Shape in the Responses of Tsetse (Glossina fuscipes and G. palpalis) to Artificial Hosts

Palpalis-group tsetse, particularly the subspecies of Glossina palpalis and G. fuscipes, are the most important transmitters of human African trypanomiasis (HAT), transmitting >95% of cases. Traps and insecticide-treated targets are used to control tsetse but more cost-effective baits might be developed through a better understanding of the fly's host-seeking behaviour. Electrocuting grids were used to assess the numbers of G. palpalis palpalis and G. fuscipes quanzensis attracted to and landing on square or oblong targets of black cloth varying in size from 0.01 m(2) to 1.0 m(2). For both species, increasing the size of a square target from 0.01 m(2) (dimensions=0.1 × 0.1 m) to 1.0 m(2) (1.0 × 1.0 m) increased the catch ~4x however the numbers of tsetse killed per unit area of target declined with target size suggesting that the most cost efficient targets are not the largest. For G. f. quanzensis, horizontal oblongs, (1 m wide × 0.5 m high) caught ~1.8x more tsetse than vertical ones (0.5 m wide × 1.0 m high) but the opposite applied for G. p. palpalis. Shape preference was consistent over the range of target sizes. For G. p. palpalis square targets caught as many tsetse as the oblong; while the evidence is less strong the same appears to apply to G. f. quanzensis. The results suggest that targets used to control G. p. palpalis and G. f. quanzensis should be square, and that the most cost-effective designs, as judged by the numbers of tsetse caught per area of target, are likely to be in the region of 0.25 × 0.25 m(2). The preference of G. p. palpalis for vertical oblongs is unique amongst tsetse species, and it is suggested that this response might be related to its anthropophagic behaviour and hence importance as a vector of HAT.     

Nitrate transport across the tonoplast of Cucumis sativus L. root cells

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO3- ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of delta pH, indicating the NO3(-)-induced proton efflux from vesicles. NO3(-)-dependent H+ efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO3-/H+ antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO3(-)-linked H+ efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO3-/H+ antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO4, significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity.     

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background

The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8⁺ and CD4⁺ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects.

Methodology/Principal Findings

To establish the basis for a SAPN-based vaccine, B- and CD8⁺ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ⁺, IL-2⁺) long-lived central memory CD8⁺ T-cells. Furthermore, we demonstrated that these Ab or CD8⁺ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites.

Conclusion

The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.     

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/-; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/- myocytes had significantly shorter action potential durations (APD) and increased steady-state (Iss) and inward rectifier (I(K1)) potassium currents compared with those of wild-type littermate cells. In Cx43+/- hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/- hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.     

The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Osmotic cell swelling activates Cl⁻ channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56^lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I_Cl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I_Cl−swell is defective in p56^lck-deficient cells. Retransfection of p56^lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I_Cl−swell. Addition of purified p56^lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56^lck washed into the cytoplasm activates I_Cl−swell in native and p56^lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56^lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56^lck. We conclude that osmotic swelling activates I_Cl−swell in lymphocytes via the tyrosine kinase p56^lck.