Malaria vectors and transmission dynamics in coastal south-western Cameroon

Background

Malaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas.

Methods

A 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months.

Results

In all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species.

Conclusion

Malaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa.

     

Cloning, E. coli expression, refolding, and ATP-binding properties of a WD40-deleted Apaf-1 isoform

The apoptotic protease activating factor (Apaf-1) is central to the regulatory mechanism by which procaspase-9 is activated in the cytochrome c-mediated pathway of apoptosis. For a detailed biochemical and structural investigation of Apaf-1 function, we have cloned and expressed in Escherichia coli inclusion bodies the WD40-deleted protein (DeltaWD40 Apaf-1) from HepG2 cell. The construct contains an N-terminal His6 tag derived from the cloning vector so that the mass of the protein and the tag together is 51,594 Da, as determined by TOF/TOF mass spectrometric analysis. An optimized refolding protocol has allowed protein recovery in highly pure form. Basic fluorescence and CD probes indicate that the refolded protein retains secondary and tertiary structures, and unfolds in the presence of higher concentration of denaturant. The equilibrium ATP binding property of the protein has been measured by changes in fluorescence emission due to the fluorescent ATP analog, mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate). The results demonstrate a tight Apaf-1-ATP interaction, the binding affinity being 380 nM.     

Complexity of aromatic ring-flip motions in proteins: Y97 ring dynamics in cytochrome c observed by cross-relaxation suppressed exchange NMR spectroscopy

Dynamics of large-amplitude conformational motions in proteins are complex and less understood, although these processes are intimately associated with structure, folding, stability, and function of proteins. Here, we use a large set of spectra obtained by cross-relaxation suppressed exchange NMR spectroscopy (EXSY) to study the 180° flipping motion of the Y97 ring of horse ferricytochrome c as a function of near-physiological temperature in the 288–308 K range. With rising temperature, the ring-flip rate constant makes a continuous transition from Arrhenius to anti-Arrhenius behavior through a narrow Arrhenius-like zone. This behavior is seen not only for the native state of the protein, but also for native-like states generated by adding subdenaturing amounts of guanidine deuterochloride (GdnDCl). Moderately destabilizing concentrations of the denaturant (1.5 M GdnDCl) completely removes the Arrhenius-like feature from the temperature window employed. The Arrhenius to anti-Arrhenius transition can be explained by the heat capacity model where temperature strengthens ground state interactions, perhaps hydrophobic in nature. The effect of the denaturant may appear to arise from direct protein-denaturant interactions that are structure-stabilizing under subdenaturing conditions. The temperature distribution of rate constants under different stability conditions also suggests that the prefactor in Arrhenius-like relations is temperature dependent. Although the use of the transition state theory (TST) offers several challenges associated with data interpretation, the present results and a consideration of others published earlier provide evidence for complexity of ring-flip dynamics in proteins.     

Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride

To find out the changes in the internal dynamics and function of proteins as a consequence of their binding interactions with guanidine hydrochloride (GdnHCl), laser flash photolysis and optical absorption methods have been used to study the dynamic events in the horse myoglobin–CO complex (MbCO) in the presence of subdenaturing concentrations of GdnHCl at pH 7, 22 °C. The rate coefficients for geminate rebinding and bimolecular rebinding (k _on) were measured by laser photolysis of CO in MbCO, and the CO dissociation rate (k _off) was determined by the CO replacement method using hexacyanoferrate ion or NO. Starting from the native-state condition, the values of k _on and k _off decrease by approximately 1.4 (±0.1)-fold in the presence of 0.1–0.3 M GdnHCl, and then increase at higher concentrations of the denaturant. This has been taken as evidence for internal motional constraints and increased stability of the protein in the subdenaturing region giving rise to gated entry of the photolyzed CO from the solvent. The rate for geminate rebinding does not show any decrease in the rate versus GdnHCl concentration plots. The values for the activation enthalpy for the CO dissociation reaction and the entropy loss relative to the native-state entropy, both measured as a function of GdnHCl concentration, indicate that the protein is indeed stabilized under subdenaturing conditions. Analyses of thermal unfolding transitions of myoglobin in the presence of different concentrations of GdnHCl indicate that the stability of this protein extracted from the linear free energy model is approximately 3–4 kcal mol⁻¹ less than the true stability. The results indicate the appropriateness of the denaturant binding model for the analysis of GdnHCl-induced unfolding data, and provide a value of 7.9 kcal mol⁻¹ as the true stability of the protein. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Colcemid effects on B16 melanoma cell progression and aberrant mitotic division

Mitotic cells selectively harvested after several h of colcemid treatment are routinely used to obtain synchronized cell cultures. DNA flow cytometry shows that when colcemid-treated B16 mitotic cells divide, they give rise to daughter cells in G1, some of which contain abnormal amounts of DNA. Two subpopulations appear to exist, one having a DNA content distribution expected of G1 cells, another having a mean DNA content about 0.8 of expected and an SD of DNA content more than 5 times expected. The effect was dependent on dose and duration of exposure to colcemid. Colcemid was more cytotoxic to cells in G2 + M than to G1 + S phase cells, and it slowed the progression of G1 cells to S. These effects of colcemid were much greater in aneuploid B16 melanoma cells than in pseudodiploid Chinese hamster ovary (CHO) cells.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation

Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.