Drug susceptibility of Plasmodium falciparum from polyclonal isolates

Msp-1 and Msp-2 genes, each present as a unique copy in the genome of Plasmodium, contain polymorphic repeats in bloc 2. We studied allelic polymorphism of Msp-1 and Msp-2 by amplifying bloc 2 with a fluorescent primer, and analysing the fragment generated. We validated this method by mixing two cloned strains: chloroquine-susceptible HB3-Honduras and chloroquine-resistant FCM29-Cameroon. This method was then used to quantify the clones in natural isolates of 19 infected persons during quinine treatment. The fragment analysis method detects efficiently clone numbers and the proportions of each in isolates.     

Glutathione transferases sequester toxic dinitrosyl-iron complexes in cells. A protection mechanism against excess nitric oxide

It is now well established that exposure of cells and tissues to nitric oxide leads to the formation of a dinitrosyl-iron complex bound to intracellular proteins, but little is known about how the complex is formed, the identity of the proteins, and the physiological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepatocytes, we here identify the complex as a dinitrosyl-diglutathionyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (GSTs) with extraordinary high affinity (K(D) = 10(-10) m). This complex is formed spontaneously through NO-mediated extraction of iron from ferritin and transferrin, in a reaction that requires only glutathione. In hepatocytes, DNDGIC may reach concentrations of 0.19 mm, apparently entirely bound to Alpha class GSTs, present in the cytosol at a concentration of about 0.3 mm. Surprisingly, about 20% of the dinitrosyl-glutathionyl-iron complex-GST is found to be associated with subcellular components, mainly the nucleus, as demonstrated in the accompanying paper (Stella, L., Pallottini, V., Moreno, S., Leoni, S., De Maria, F., Turella, P., Federici, G., Fabrini, R., Dawood, K. F., Lo Bello, M., Pedersen, J. Z., and Ricci, G. (2007) J. Biol. Chem. 282, 6372-6379). DNDGIC is a potent irreversible inhibitor of glutathione reductase, but the strong complex-GST interaction ensures full protection of glutathione reductase activity in the cells, and in vitro experiments show that damage to the reductase only occurs when the DNDGIC concentration exceeds the binding capacity of the intracellular GST pool. Because Pi class GSTs may exert a similar role in other cell types, we suggest that specific sequestering of DNDGIC by GSTs is a physiological protective mechanism operating in conditions of excessive levels of nitric oxide.     

Electrostatic association of glutathione transferase to the nuclear membrane. Evidence of an enzyme defense barrier at the nuclear envelope

The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl-iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364-6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.     

IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation

Previous work from our laboratory has shown that human ether à go-go (hEAG) K(+) channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin-like growth factor-1 (IGF-1), which is known to stimulate cell proliferation. Acute applications of IGF-1 increased K(+) current-density and hyperpolarized MCF-7 cells. The effects of IGF-1 were inhibited by hEAG inhibitors. Moreover, IGF-1 increased mRNA expression of hEAG in a time-dependent manner in parallel with an enhancement of cell proliferation. The MCF-7 cell proliferation induced by IGF-1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) are known to mediate IGF-1 cell proliferative signals through the activation of extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF-7 cells, IGF-1 rapidly stimulated Akt phosphorylation, whereas IGF-1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF-1 on K(+) current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF-1 increases both the activity and the expression of hEAG channels through an Akt-dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF-1 may thus play an important role in IGF-1 signaling to promote a mitogenic effect in breast cancer cells.     

Epac- and Rap- independent ERK1/2 phosphorylation induced by Gs-coupled receptor stimulation in HEK293 cells

Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.     

Incidence,Carriage and Case-Carrier Ratios for Meningococcal Meningitis in the African Meningitis Belt: A Systematic Review and Meta-Analysis

BackgroundTo facilitate the interpretation of meningococcal meningitis epidemiology in the “African meningitis belt”, we aimed at obtaining serogroup-specific pooled estimates of incidence, carriage and case-carrier ratios for meningococcal meningitis in the African meningitis belt and describe their variations across the endemic, hyperendemic and epidemic context.MethodsWe conducted a systematic review and meta-analysis of studies reporting serogroup-specific meningococcal meningitis monthly incidence and carriage in the same population and time period. Epidemiological contexts were defined as endemic (wet season, no epidemic), hyperendemic (dry season, no epidemic), and epidemic (dry season, epidemic).FindingsEight studies reporting a total of eighty pairs of serogroup-specific meningococcal meningitis incidence and carriage estimates were included in this review. For serogroup A, changes associated with the transition from endemic to hyperendemic incidence and from hyperendemic to epidemic incidence were 15-fold and 120-fold respectively. Changes in carriage prevalence associated with both transitions were 1-fold and 30-fold respectively.
For serogroup W and X, the transition from endemic to hyperendemic incidence involved a 4-fold and 1•1-fold increase respectively. Increases in carriage prevalence for the later transition were 7-fold and 1•7-fold respectively. No data were available for the hyperendemic-epidemic transition for these serogroups. Our findings suggested that the regular seasonal variation in serogroup A meningococcal meningitis incidence between the rainy and the dry season could be mainly driven by seasonal change in the ratio of clinical cases to subclinical infections. In contrast appearance of epidemic incidences is related to a substantial increase in transmission and colonisation and to lesser extent with changes in the case-carrier ratio.ConclusionSeasonal change in the rate of progression to disease given carriage together with variations in frequency of carriage transmission should be considered in models attempting to capture the epidemiology of meningococcal meningitis and mainly to predict meningitis epidemics in the African meningitis belt.     

Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops

The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA （RAPD） technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.     

Estimating the Disease Burden of Pandemic (H1N1) 2009 Virus Infection in Hunter New England,Northern New South Wales,Australia, 2009

Introduction

On May 26, 2009, the first confirmed case of Pandemic (H1N1) 2009 virus (pH1N1) infection in Hunter New England (HNE), New South Wales (NSW), Australia (population 866,000) was identified. We used local surveillance data to estimate pH1N1-associated disease burden during the first wave of pH1N1 circulation in HNE.

Methods

Surveillance was established during June 1-August 30, 2009, for: 1) laboratory detection of pH1N1 at HNE and NSW laboratories, 2) pH1N1 community influenza-like illness (ILI) using an internet survey of HNE residents, and 3) pH1N1-associated hospitalizations and deaths using respiratory illness International Classification of Diseases 10 codes at 35 HNE hospitals and mandatory reporting of confirmed pH1N1-associated hospitalizations and deaths to the public health service. The proportion of pH1N1 positive specimens was applied to estimates of ILI, hospitalizations, and deaths to estimate disease burden.

Results

Of 34,177 specimens tested at NSW laboratories, 4,094 (12%) were pH1N1 positive. Of 1,881 specimens from patients evaluated in emergency departments and/or hospitalized, 524 (26%) were pH1N1 positive. The estimated number of persons with pH1N1-associated ILI in the HNE region was 53,383 (range 37,828–70,597) suggesting a 6.2% attack rate (range 4.4–8.2%). An estimated 509 pH1N1-associated hospitalizations (range 388–630) occurred (reported: 184), and up to 10 pH1N1-associated deaths (range 8–13) occurred (reported: 5). The estimated case hospitalization ratio was 1% and case fatality ratio was 0.02%.

Discussion

The first wave of pH1N1 activity in HNE resulted in symptomatic infection in a small proportion of the population, and the number of HNE pH1N1-associated hospitalizations and deaths is likely higher than officially reported.     

Bioinformatics and data mining in proteomics

Proteomic studies involve the identification as well as qualitative and quantitative comparison of proteins expressed under different conditions, and elucidation of their properties and functions, usually in a large-scale, high-throughput format. The high dimensionality of data generated from these studies will require the development of improved bioinformatics tools and data-mining approaches for efficient and accurate data analysis of biological specimens from healthy and diseased individuals. Mining large proteomics data sets provides a better understanding of the complexities between the normal and abnormal cell proteome of various biological systems, including environmental hazards, infectious agents (bioterrorism) and cancers. This review will shed light on recent developments in bioinformatics and data-mining approaches, and their limitations when applied to proteomics data sets, in order to strengthen the interdependence between proteomic technologies and bioinformatics tools.     

Modulation of dendritic cell function by naive and regulatory CD4+ T cells

The consequences of interactions between dendric cells (DCs) and either naive CD4+ T cells or regulatory CD4+CD25+ T cells on the expression of proinflammatory IL-6 and anti-inflammatory IL-10 in DC were examined over a period of 12 h, spanning the time frame during which stable T cell-DC interactions shape the development of tolerance and immunity in vivo. We demonstrate that the basal production of IL-6 and IL-10, which is initiated following DC stimulation with LPS, is modified in distinctly different ways by interaction with the two T cell populations. Naive CD4 T cells skew DC cytokine production toward IL-6 and suppress IL-10, whereas CD4+CD25+ T cells have the opposite effect. CD8 T cells or memory CD4 T cells do not influence basal cytokine production by stimulated DC. The effect of CD4+CD25+ T cells is dominant in coculture with naive CD4 T cells as long as inflammatory LPS is absent; the addition of LPS abrogates the suppression of IL-6. However, the modulating influence of CD4+CD25+ T cells remains evident in the enhancement of IL-10 production. Thus, mutual interactions between DC and CD4+ T cell subpopulations following contact with pathogens are likely to influence the strength and quality of incipient immune responses in the local microenvironment.