Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Biochemical and genetic analysis of the vaccinia virus d5 protein: Multimerization-dependent ATPase activity is required to support viral DNA replication

The vaccinia virus-encoded D5 protein is an essential ATPase involved in viral DNA replication. We have expanded the genotypic and phenotypic analysis of six temperature-sensitive (ts) D5 mutants (Cts17, Cts24, Ets69, Dts6389 [also referred to as Dts38], Dts12, and Dts56) and shown that at nonpermissive temperature all of the tsD5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For Cts17 and Cts24, this restriction reflects the thermolability of the D5 proteins. The Dts6389, Dts12, and Dts56 D5 proteins become insoluble at 39.7 degrees C, while the Ets69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7 degrees C. To investigate which structural features of D5 are important for its biological and biochemical activities, we generated targeted mutations in invariant residues positioned within conserved domains found within D5. Using a transient complementation assay that assessed the ability of D5 variants to sustain ongoing DNA synthesis during nonpermissive Cts24 infections, only a wtD5 allele supported DNA synthesis. Alleles of D5 containing targeted mutations within the Walker A or B domains, the superfamily III helicase motif C, or the AAA+ motif lacked biological competency. Furthermore, purified preparations of these variant proteins revealed that they all were defective in ATP hydrolysis. Multimerization of D5 appeared to be a prerequisite for enzymatic activity and required the Walker B domain, the AAA+ motif, and a region located upstream of the catalytic core. Finally, although multimerization and enzymatic activity are necessary for the biological competence of D5, they are not sufficient.     

Gender-influenced obesity QTLs identified in a cross involving the KK type II diabetes-prone mouse strain

The inheritance of adiposity and related traits has been investigated in the obese, diabetes-prone KK/HlLt (KK) and the lean, normoglycemic C57BL/6J (B6) mouse strains, their F₁ hybrids, and a large intercross generation. Adiposity index (AI) was defined as the sum of four fat depot weights divided by body weight. Both male and female KK mice were obese, but AI values averaged twofold higher in females than in males. In contrast, B6 females were slightly more lean than males. A genome-wide search revealed several qualitative trait loci (QTLs) affecting AI. The proximal region of Chromosome (Chr) 9 has a large effect on AI, with a much stronger effect in females (lod = 6.3) than in males (lod = 2.7). The data for females fit a model in which a dominant allele from KK increases AI by 30%, with the lod score peak falling between markers D9Mit66 and D9Mit328. This QTL has large effects on inguinal and mesenteric fat pad weights, with smaller effects on gonadal and retroperitoneal fat pads. The region of Chr 9 containing this QTL has extensive homology to human Chr 11q. An X-linked QTL affecting AI was evident in males (lod = 3.77), but not females (lod = 0.7). Exclusion of mesenteric fat from male AI resulted in an increased lod score (lod = 5.0) at 8 cM distal to DXMit166. A suggestive AI QTL (lod = 4.2), differentially affecting males, was localized to Chr 18 near the glucocorticoid receptor locus. A region of Chr 7 had a strong effect on body weight (lod = 6.9), a significant effect on inguinal fat% (lod = 4.4), and a suggestive effect on AI in females (lod = 4.1). Plasma leptin levels were associated with genotypes on Chr 9 (lod = 5.9) and Chr 7 (lod = 4.2). A region of Chr 1 had a suggestive effect on fasted blood glucose (lod = 3.6). Received: 23 March 1999 / Accepted: 2 June 1999     

Theoretical study on temperature dependence of cellular uptake of QDs nanoparticles

Cellular uptake kinetics of nanoparticles is one of the key issues determining the design and application of the particles. Models describing nanoparticles intrusion into the cell mostly take the endocytosis process into consideration, and the influences of electrical charges, sizes, concentrations of the particles have been investigated. In this paper, the temperature effect on the cellular uptake of Quantum Dots (QDs) is studied experimentally. QDs are incubated with the SPCA-1 human lung tumor cells, and the nanoparticles on the cell membrane and inside the cell are quantified according to the fluorescence intensities recorded. It is found that the amounts of nanoparticles attached onto the cell membrane and inside the cell both increase with temperature. Based on the experimental results, a model is proposed to describe the cellular uptake dynamic process of nanoparticles. The process consists of two steps: nanoparticles adsorption onto the cell membrane and the internalization. The dynamic parameters are obtained through curve fitting. The simulated results show that the internalization process can be categorized into different phases. The temperature dependent internalization rate constant is very small when below 14?°C. It increases distinctly when temperature rises from 14?°C to 22?°C, but there is no evident increase as temperature further increases above 22?°C. Results show that by incorporating a temperature-independent internalization factor, the model predictions well fit the experimental results.     

HIV/AIDS research conducted in the developing world and sponsored by the developed world: reporting of research ethics committee review in two countries

We explored how often journal articles reporting HIV research sponsored by a developed country, but conducted in a developing country, mention research ethics committee (REC) approval from both countries, and what factors are involved. Of all such 2007 articles on Medline conducted in one of four developing countries (N = 154), only 52% mentioned such dual approval. Mention of dual vs. single approval was more likely among articles with ≥ 50% sponsor country authors, and the United States as the sponsor country. Also, dual approval was more likely among articles that mentioned informed consent and funding, had ≥ 50% sponsor country authors, were biomedical (vs. psychosocial), and appeared in journals adopting International Committee Medical Journal Editors (ICMJE) guidelines. Dual approval was thus obtained in only half of the articles and was associated with ethical and logistic issues, indicating the need for clearer and more universally accepted guidelines.     

Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.     

Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.