The accumulation of lactic acid and its influence on the growth ofPlasmodium falciparum in synchronized cultures

Summary Synchronization ofPlasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66±0.55 μmol LA per 12 h per 10⁷ parasitized cells (n=26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72±0.62 μmol LA per 12 hours per 10⁹ red blood cells) (n=41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth ofPlasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield. J. W. Z. was supported during part of this study by a long-term fellowship of the European Molecular Biology Organization, Heidelberg, West Germany, followed by a Research Associateship from the National Research Council, Washington, D.C. The project was supported by grants from the Medical Research Council to J. G. S. and by the Naval Research and Development Command, Work Unit No. 3M 162 770 A871 AE 312. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the naval service at large.     

Specific haptoglobin expression in bronchoalveolar lavage during differentiation of circulating fibroblast progenitor cells in mild asthma

Haptoglobin is an acute-phase glycoprotein considered to be involved in tissue repair and is produced by fibroblasts and inflammatory cells. By using a gel-based proteomic approach, we show for the first time a possible role for haptoglobin in the differentiation of fibroblast progenitor cells, termed fibrocytes, in patients with mild asthma. Bronchoalveolar lavage fluid (BALF) was performed to sample circulating fibrocytes from patients with mild asthma and nonasthmatic control subjects. Fibrocytes from the airway lumen were characterized by triple staining of the markers CD34/CD45R0/alpha-smooth muscle actin, and subjected to confocal microscopy. The protein expression pattern was analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). Elevated levels of haptoglobin expression in BALF was reported in a sub-group of patients with mild asthma (p < 0.05) when compared to the other subjects. In addition, this increase in haptoglobin was accompanied by differentiation of fibrocytes into fibroblast-like cells. When further analyzing the expression pattern of haptoglobin isoforms, a heterozygous expression was detected in the patients where fibrocyte differentiation could be observed. These data raise the possibility that an acute and specific inflammatory state facilitates the differentiation of fibroblast progenitor cells into activated fibroblasts. Furthermore, this study proposes a novel role for haptoglobin in airway remodeling in patients with asthma.     

Attributions of Cancer ‘Alarm’ Symptoms in a Community Sample

BackgroundAttribution of early cancer symptoms to a non-serious cause may lead to longer diagnostic intervals. We investigated attributions of potential cancer ‘alarm’ and non-alarm symptoms experienced in everyday life in a community sample of adults, without mention of a cancer context.MethodsA questionnaire was mailed to 4858 adults (≥50 years old, no cancer diagnosis) through primary care, asking about symptom experiences in the past 3 months. The word cancer was not mentioned. Target ''alarm'' symptoms, publicised by Cancer Research UK, were embedded in a longer symptom list. For each symptom experienced, respondents were asked for their attribution (‘what do you think caused it''), concern about seriousness (‘not at all’ to ‘extremely’), and help-seeking (‘did you contact a doctor about it’: Yes/No).ResultsThe response rate was 35% (n = 1724). Over half the respondents (915/1724; 53%) had experienced an ‘alarm’ symptom, and 20 (2%) cited cancer as a possible cause. Cancer attributions were highest for ‘unexplained lump’; 7% (6/87). Cancer attributions were lowest for ‘unexplained weight loss’ (0/47). A higher proportion (375/1638; 23%) were concerned their symptom might be ‘serious’, ranging from 12% (13/112) for change in a mole to 41% (100/247) for unexplained pain. Just over half had contacted their doctor about their symptom (59%), although this varied by symptom. Alarm symptoms were appraised as more serious than non-alarm symptoms, and were more likely to trigger help-seeking.ConclusionsConsistent with retrospective reports from cancer patients, ‘alarm’ symptoms experienced in daily life were rarely attributed to cancer. These results have implications for understanding how people appraise and act on symptoms that could be early warning signs of cancer.     

Biphasic regulation of adenylate cyclase by cholera toxin in neuroblastoma x glioma hybrid cells is due to the activation and subsequent loss of the alpha subunit of the stimulatory GTP binding protein (GS)

Exposure of neuroblastoma x glioma hybrid (NG108-15) cells to low concentrations of cholera toxin produced a stimulation of both basal and forskolin-amplified adenylate cyclase activity in membranes prepared from these cells. Higher concentrations of cholera-toxin reversed this effect. Mn2+ activation of adenylate cyclase indicated that this effect was not due to a modification of the intrinsic activity of this enzyme. Cholera toxin was demonstrated to produce a concentration and time-dependent loss of GS alpha from membranes of these cells. Loss of GS alpha from membranes of these cells was preceded by its ADP-ribosylation. The effects of cholera toxin were specific for GS alpha, as no alterations in levels of the pertussis toxin-sensitive G-proteins Gi2, Gi3 and Go, were noted in parallel. Equally, no alteration in levels of G-protein beta-subunit were produced by the cholera toxin treatment. These experiments demonstrate that cholera toxin-catalysed ADP-ribosylation does not simply maintain an activated population of GS at the plasma membrane and that alterations in levels of GS at the plasma membrane can modify adenylate cyclase activity.     

The frequency and duration of Salmonella–macrophage adhesion events determines infection efficiency

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.     

Distribution of lipopolysaccharide and the detection of a new subfraction in the cell envelope of a marine pseudomonad.

The three outer layers of the cell envelope of marine pseudomonad B-16, the loosely bound outer layer, the outer membrane, and the periplasmic space layer, are the only ones containing appreciable amounts of both lipid and carbohydrate. These layers and a fraction released into the medium during growth of the cells were examined for the presence of common antigens by double immunodiffusion using anti-whole serum. Each of the layers, the medium fraction, and lipopolysaccharide (LPS) isolated from the organism were shown to contain two or more diffusible components showing reactions of identity. Thus LPS is found in each of the three outer layers of the cell envelope of this gram-negative bacterium. The periplasmic space layer was found to contain a fraction accounting for 20% of the dry weight of the layer, which was sedimentable at 30,000 x g and contained lipid, protein, and carbohydrate. Double-immunodiffusion tests indicated that the fraction contained at least one of the two antigens present in isolated LPS. A particulate material was released by the cells during growth which gave a positive test for 2-keto-3-deoxyoctulosonic acid and cross-reacted serologically with LPS.     

Targeting of Arenavirus RNA Synthesis by a Carboxamide-Derivatized Aromatic Disulfide with Virucidal Activity

Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37°C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed.     

A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA

About 1% of the mouse genome is cleaved by Hpa II to give a discrete fraction on gels. The nonmethylated fraction is present in all tested tissues, including sperm, and contains Hpa II sites at about 15 times their frequency in bulk DNA. About 80% of the fraction is composed of sequences that occur once or a few times per genome; the remainder is largely rDNA. Unlike bulk DNA, the fraction is not deficient in CpG, and this may be directly due to the lack of methylation. Genomic mapping of three nonribosomal fragments showed that they are part of islands of DNA within which nonmethylated Hpa II and Hha I sites are highly concentrated. We estimate about 30,000 islands per haploid genome and discuss evidence that many may be associated with genes.