Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.     

What spectroscopy can still tell us about the secondary structure of bacteriorhodopsin.

The recently published model of the structure of bacteriorhodopsin (bR), developed by fitting the peptide chain to a high-resolution, three-dimensional density map, rules out the existence of transmembrane beta-sheet and provides an accurate estimate of the helix content. The precise geometry of the dihedral angles in the helical regions of the polypeptide cannot yet be specified from the diffraction data, however. Published data on the circular dichroism (CD) spectrum between 190 and 240 nm, and the infrared (IR) spectrum in the amide I band suggest that the helical conformation in bR may be, for the most part, a rather unusual one. The precise structural model, which specifies the number of residues in transmembrane helices, can now be used as an additional constraint in seeking models of the helical conformation that are in quantitative agreement with the CD and IR spectroscopic data. Further spectroscopic measurements can also be used to determine whether there are changes in the unusual dihedral-angle conformation within the helices during the photocycle.     

The Development of Indonesian Online Game Addiction Questionnaire

Online game is an increasingly popular source of entertainment for all ages, with relatively prevalent negative consequences. Addiction is a problem that has received much attention. This research aims to develop a measure of online game addiction for Indonesian children and adolescents. The Indonesian Online Game Addiction Questionnaire draws from earlier theories and research on the internet and game addiction. Its construction is further enriched by including findings from qualitative interviews and field observation to ensure appropriate expression of the items. The measure consists of 7 items with a 5-point Likert Scale. It is validated by testing 1,477 Indonesian junior and senior high school students from several schools in Manado, Medan, Pontianak, and Yogyakarta. The validation evidence is shown by item-total correlation and criterion validity. The Indonesian Online Game Addiction Questionnaire has good item-total correlation (ranging from 0.29 to 0.55) and acceptable reliability (α = 0.73). It is also moderately correlated with the participant''s longest time record to play online games (r = 0.39; p<0.01), average days per week in playing online games (ρ = 0.43; p<0.01), average hours per days in playing online games (ρ = 0.41; p<0.01), and monthly expenditure for online games (ρ = 0.30; p<0.01). Furthermore, we created a clinical cut-off estimate by combining criteria and population norm. The clinical cut-off estimate showed that the score of 14 to 21 may indicate mild online game addiction, and the score of 22 and above may indicate online game addiction. Overall, the result shows that Indonesian Online Game Addiction Questionnaire has sufficient psychometric property for research use, as well as limited clinical application.     

In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection.     

Insulin-like growth factor-1 increases endothelin receptor A levels and action in cultured rat aortic smooth muscle cells

Insulin is known to cause an increase in endothelin-1 (ET-1) receptors in vascular smooth muscle cells (SMCs), but the effect of insulin-like growth factor 1 (IGF-1) on ET-1 receptor expression is not known. We therefore carried out the present study to determine the effect of IGF-1 on the binding of ET-1 to, and ET type A receptor (ETAR) expression and ET-1-induced 3H-thymidine incorporation in, vascular SMCs. In serum-free medium, IGF-1 treatment increased the binding of 125I-ET-1 to SMC cell surface ET receptors from a specific binding of 20.1%+/-3.1% per mg of protein in control cells to 45.1%+/-8.6% per mg of protein in cells treated with IGF-1 (10 nM). The effect of IGF-1 was dose-related, with a significant effect (1.4-fold) being seen at 1 nM. The minimal time for IGF-1 treatment to be effective was 30 min and the maximal effect was reached at 6 h. Immunoblotting analysis showed that ETAR expression in IGF-1-treated cells was increased by 1.7-fold compared to controls. Levels of ETAR mRNA measured by the RT-PCR method and Northern blotting were also increased by 2-fold in IGF-1-treated SMCs. These effects of IGF-1 were abolished by cycloheximide or genistein. Finally, ET-1-stimulated thymidine uptake and cell proliferation were enhanced by IGF-1 treatment, with a maximal increase of 3.2-fold compared to controls. In conclusion, in vascular SMCs, IGF-1 increases the expression of the ET-1 receptor in a dose- and time-related manner. This effect is associated with increased thymidine uptake and involves tyrosine kinase activation and new protein synthesis. These findings support the role of IGF-1 in the development of atherosclerotic, hypertensive, and diabetic vascular complications.     

Changing tumour antigen expression in metastatic hepatocellular carcinoma cells of the guinea pig

Hepatocellular carcinoma cells (Line-10), obtained from ascitic fluid after diethylnitrosamine treatment of Sewall Wright strain-2 guinea pigs, produce solid (primary) tumours, lymph-node and lung metastases and malignant ascites when reinjected into animals of the same strain. Monoclonal antibodies were raised against the tumour cells by immunizing BALB/c mice with viable ascitic hepatocellular Line-10 tumour cells. Three hybridomas producing anti-Line-10 monoclonal antibodies were selected for further studies (10TL1, 10TL40 and 10TL43) and compared with monoclonal antibodies against intermediate filament keratins. The anti-Line-10 monoclonal antibodies did not cross react with Line-1 hepatocellular carcinoma cells, nor with normal guinea pig hepatocytes. When ascitic Line-10 cells form high papillary projections on the peritoneal surface, they significantly reduced their antigen expression of 10TL40 and of 10TL43 defined antigens, while the expression of 10TL1 defined antigens remained unaltered. Invading Line-10 cells in the deep submesothelial stromal tissue, however, lost reactivity with MoAb 10TL43 but not with the MoAb's 10TL40 and 10TL1. The antigens on lung- and lymphnode metastases remained largely unaffected. The reactivity with MoAb's 10TL40 and with 10TL43 was also lost upon prolonged culturing of Line-10 cells. The reactivity of Line-10 and Line-1 cells with all monoclonal antibodies against keratin filaments remained unaltered. Line-1 cells could be distinguished from Line-10 cells by the absence of any reactivity with the MoAb's 10TL1, -40, -43, but also by the fact that 100% of the Line-1 cells were positive with antibodies against keratins 5/8, 18, and 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional electron diffraction of PhoE porin to 2.8 A resolution

A three-dimensional set of electron diffraction intensities of PhoE porin embedded in trehalose extending to 2.8 A resolution has been collected and analyzed. The strongest high-resolution intensities are distributed as a figure of revolution about the z*-axis and are located primarily in a resolution range of 4.5 A to 5.0 A. Within this region, centered near 4.8 A resolution the brightest intensities are clustered about inclination angles of 35 degrees and 0 degrees from the a*, b* plane. This distribution of intensities indicates that the beta-sheet in PhoE porin is arranged to form a cylinder-like structure that contains major populations of beta-sheet strands tilted an average of 35 degrees and 0 degrees with respect to the membrane plane normal. This cylindrical structure has been seen previously in the high-resolution projection map of PhoE as an elliptical ring of high density.     

Water transport in AQP0 aquaporin: molecular dynamics studies

A double lipid bilayer structure containing opposing tetramers of AQP0 aquaporin, in contact through extracellular face loop regions, was recently modeled using an intermediate-resolution map obtained by electron crystallographic methods. The pores of these water channels were found to be critically narrow in three regions and subsequently interpreted to be those of a closed state of the channel. The subsequent determination of a high-resolution AQP0 tetramer structure by X-ray crystallographic methods yielded a pore model featuring two of the three constrictions as noted in the EM work and water molecules within the channel pore. The extracellular-side constriction region of this AQP0 structure was significantly larger than that of the EM-based model and similar to that of the highly water permeable AQP1. The X-ray-based study of AQP0 however could not ascertain if the water molecules found in the pore were the result of water entering from one or both ends of the channel, nor whether water could freely pass through all constriction points. Additionally, this X-ray-based structure could not provide an answer to the question of whether the double lipid bilayer configuration of AQP0 could functionally maintain a water impermeable state of the channel. To address these questions we conducted molecular dynamics simulations to compare the time-dependent behavior of the AQP0 and AQP1 channels within lipid bilayers. The simulations demonstrate that AQP0, in single or double lipid bilayers, is not closed to water transport and that thermal motions of critical side-chains are sufficient to facilitate the movement of water past any of its constriction regions. These motional requirements do however lead to significant free energy barriers and help explain physiological observations that found water permeability in AQP0 to be substantially lower than in the AQP1 pore.