Neurological symptoms in tricolor sharkminnow Balantiocheilos melanopterus associated with Myxobolus balantiocheili n. sp. infecting the central nervous system

A new species, Myxobolus balantiocheili, is described from the central nervous system of wild and cultured tricolor sharkminnow Balantiocheilos melanopterus from Thailand. The infection was associated with emaciation and severe neurological symptoms, including sudden uncoordinated darting, rolling and pitching. Whitish cysts of varying shape, measuring 70 to 500 microm, were located in the brain, spinal cord and in the meninx, and were sometimes accompanied by haemorrhages. In the brain, most cysts were located in the medulla oblongata and the mesencephalon. Cysts with a segmental distribution were observed in the meninx of the spinal cord. Single di- to hexasporic pansporoblasts, and free mature spores, were frequently seen in both the white and grey matter of the brain and spinal cord, mostly associated with blood vessels. Mature spores varied in shape from oval to ellipsoid, measuring 12.3 +/- 0.59 (range 10.6 to 13.3) x 10.0 +/- 0.47 (9.2 to 10.9) x 6.9 +/- 0.20 (6.7 to 7.1) microm. Spore size and morphology, in addition to fish-host species and apparently narrow tissue tropism, clearly distinguishes this new Myxobolus. No marked inflammatory response or gliosis was associated with the lesions, but compression and degeneration of the nervous tissue surrounding large cysts was observed. We suggest that the high temperature of the fish hosts' tropical habitat may have increased the rate of development and the virulence of M. balantiocheili.     

The presence of promatrix metalloproteinase-3 and its relation with different categories of coal workers' pneumoconiosis

Extracellular matrix formation (ECM) and remodeling are critical events related to the pathogenesis of pulmonary fibrosis. Matrix metalloproteinases play an essential role in degrading and remodeling the ECM. In this study, we tried to show the presence and correlation of promatrix metalloproteinase-3 (proMMP-3) (the inactive form of metalloproteinase-3) levels in coal workers' pneumoconiosis (CWP) with different categories. The study population consisted of 44 coal miners with CWP (pos CWP). Coal miners without CWP (neg CWP, n = 24) and non-underground personnel (controls, n = 17) were taken as controls. All coal miners were stable and had no systemic infection or disease. Standard posterio-anterior chest radiographs and pulmonary function tests were performed to exclude any diseases other than CWP. Serum proMMP-3 was analysed using the sandwich enzyme-linked immunosorbent assay according to the manufacturer's instructions (The Binding Site, Birmingham, UK). Mean proMMP-3 values of the all three groups were compared and a significant statistical difference obtained (p < 0.001). In addition, a statistically significant difference was found between categories of the disease and proMMP-3 values (p < 0.05). The effects of age, exposure duration and cigarette smoking on proMMP-3 values in coal miners with CWP were investigated. There were no correlations between age, smoking and proMMP-3 values. However, a positive correlation was found between exposure duration and proMMP-3 values (r = 0.447, p = 0.008). In conclusion, proMMP-3 (prostromelysin 1) may play an essential role in degrading and remodeling the ECM in workers with pneumoconiosis. ProMMP-3 may also reflect the stage of pneumoconiosis disease.     

The role of TGF beta signaling in the formation of the dorsal nervous system is conserved between Drosophila and chordates

Transforming growth factor beta signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. Here, we show that the dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor beta signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes.     

Debating ethics and public policy: the Nuffield Council on Bioethics

Concerns about bioethical issues raised by scientific developments in genetics are a common feature of both public and policy debates in the UK. This article sets out how one such body, the Nuffield Council on Bioethics, contributes to public and policy debates, and provides information about similar bodies in the UK and around the world.     

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide

A fragment of the amyloid beta protein, βA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that βA(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, βA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by βA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to βA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 • - ) - and hydroxyl ( •OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of •OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of •OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to βA(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that βA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 • - can lead to HOCl and further formation of •OH, which both have a cytotoxic potential.     

Protein sorting into multivesicular endosomes

Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

Diarrhea,Stimulation and Growth Predict Neurodevelopment in Young North Indian Children

Background and Objective

Infants and young children in low to middle-income countries are at risk for adverse neurodevelopment due to multiple risk factors. In this study, we sought to identify stimulation and learning opportunities, growth, and burden of respiratory infections and diarrhea as predictors for neurodevelopment.

Methods

We visited 422 North Indian children 6 to 30 months old weekly for six months. Childhood illnesses were assessed biweekly. At end study, we assessed neurodevelopment using the Ages and Stages Questionnaire 3^rd ed. (ASQ-3) and gathered information on stimulation and learning opportunities. We identified predictors for ASQ-3 scores in multiple linear and logistic regression models.

Results

We were able to explain 30.5% of the variation in the total ASQ-3 score by the identified predictors. When adjusting for child characteristics and annual family income, stimulation and learning opportunities explained most of the variation by 25.1%. Height for age (standardized beta: 0.12, p<.05) and weight for height z-scores (std. beta: 0.09, p<.05) were positively associated with the total ASQ-3 score, while number of days with diarrhea was negatively associated with these scores (std. beta: -0.13, p<0.01).

Conclusion

Our results support the importance of early child stimulation and general nutrition for child development. Our study also suggests that diarrhea is an additional risk factor for adverse neurodevelopment in vulnerable children.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

DNA methylation abnormalities in congenital heart disease

Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.