α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

Phylogeography of a pan-Atlantic abyssal protobranch bivalve: implications for evolution in the Deep Atlantic

The deep sea is a vast and essentially continuous environment with few obvious barriers to gene flow. How populations diverge and new species form in this remote ecosystem is poorly understood. Phylogeographical analyses have begun to provide some insight into evolutionary processes at bathyal depths (<3000 m), but much less is known about evolution in the more extensive abyssal regions (>3000 m). Here, we quantify geographical and bathymetric patterns of genetic variation (16S rRNA mitochondrial gene) in the protobranch bivalve Ledella ultima, which is one of the most abundant abyssal protobranchs in the Atlantic with a broad bathymetric and geographical distribution. We found virtually no genetic divergence within basins and only modest divergence among eight Atlantic basins. Levels of population divergence among basins were related to geographical distance and were greater in the South Atlantic than in the North Atlantic. Ocean‐wide patterns of genetic variation indicate basin‐wide divergence that exceeds what others have found for abyssal organisms, but considerably less than bathyal protobranchs across similar geographical scales. Populations on either side of the Mid‐Atlantic Ridge in the North Atlantic differed, suggesting the Ridge might impede gene flow at abyssal depths. Our results indicate that abyssal populations might be quite large (cosmopolitan), exhibit only modest genetic structure and probably provide little potential for the formation of new species.     

Expression and regulation of inducible IkappaB kinase (IKK-i) in human fibroblast-like synoviocytes

IkappaB kinase (IKK) plays a key role in the regulation of nuclear factor kappaB (NF-kappaB). We previously demonstrated the expression of two kinases, IKK1 and IKK2, in fibroblast-like synoviocytes (FLS) and determined their functional consequences for inflammatory gene expression in vitro and in vivo. Recently, a novel inducible IkappaB kinase has been described, namely, IKK-i or IKK-epsilon, which is functionally and structurally distinct from constitutively expressed IKK1 and IKK2. Therefore, we investigated the expression and regulation of this novel kinase in FLS from patients with rheumatoid arthritis and osteoarthritis. Interestingly, constitutive gene expression and protein expression were observed in all cell lines examined. TNFalpha stimulation for 24 h increased IKK-i expression 7.2 +/- 1.8-fold in FLS (P < 0.02). IL-1 also significantly increased IKK-i gene expression. Time course experiments demonstrated that IKK-i gene expression increased within 3 h of TNFalpha stimulation and persisted for at least 24 h. Dose-response studies showed that as little as 1 ng/ml of TNFalpha increased IKK-i gene expression. Constitutive IKK-1 gene expression was also noted in rheumatoid arthritis, osteoarthritis, and normal synovium. This is the first report demonstrating constitutive expression and cytokine regulation of this novel kinase in primary human synovial cells.     

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.     

The formation of migratory ripples in a mixed species bacterial biofilm growing in turbulent flow

Mixed-species biofilms, consisting of Klebsiella pneumoniae , Pseudomonas aeruginosa , Pseudomonas fluorescens and Stenotrophomonas maltophilia , were grown in glass flow cells under either laminar or turbulent flow. The biofilms grown in laminar flow consisted of roughly circular-shaped microcolonies separated by water channels. In contrast, biofilm microcolonies grown in turbulent flow were elongated in the downstream direction, forming filamentous 'streamers'. Moreover, biofilms growing in turbulent flow developed extensive patches of ripple-like structures between 9 and 13 days of growth. Using time-lapse microscopic imaging, we discovered that the biofilm ripples migrated downstream. The morphology and the migration velocity of the ripples varied with short-term changes in the bulk liquid flow velocity. The ripples had a maximum migration velocity of 800 μm h⁻¹ (2.2 × 10⁻⁷ m s⁻¹) when the liquid flow velocity was 0.5 m s⁻¹ (Reynolds number = 1800). This work challenges the commonly held assumption that biofilm structures remain at the same location on a surface until they eventually detach.     

The investigation of cytosolic phospholipase A2 using ELISA

Two studies of the behaviour of cytosolic phospholipase A(2) (cPLA(2)) in the red blood cell (RBC), as measured by ELISA, are described. In the first study we show a significant increase in cPLA(2) in patients with schizophrenia compared to controls and suggest that this measure, if corroborated, could be used as a diagnostic marker. In a second study we found that washing the RBC introduced an unknown confounding variable which led us to reject this study. A subsequent investigation of washing red cells showed that the washing effect may be due to a plasma factor likely to be more than 5kDa MW which can be removed from red cells by washing with buffers. When the cells are washed, the concentration of cPLA(2) in the red cell, as measured by ELISA, significantly increases. We advise against washing the red cell in any study that involves measuring cPLA(2) by ELISA.     

Influence of roots and climate on mineral and trace element storage and flux in tropical mangrove soils

The storage and flux of various mineral and trace elements in soils (0–30cm depth) were examined in relation to monsoonal rains and fine root biomass in four mangrove forests of different age and type in southern Thailand. The onset of the wet SW monsoon resulted in the percolation and dilution of porewater solutes by rainwater and by less saline tidal water, as indicated by shifts in Eh, pH and porewater SO₄/Cl ratios. This is contrary to temperate intertidal environments where seasonal patterns of porewater constituents, and biological and biogeochemical activities, are strongly cued to temperature. Fluxes across the soil–water interface were most often not statistically significant. Concentration of dissolved porewater metals were dominated by Fe, Mn, Al, Mo and Zn. The decreasing order of solid-phase element inventories in these soils, on average, was: Al, S, Fe, Na, Mg, K, Ca, N, P, Mn, V, Zn, Cr, Ni, As, Co, Cu, Pb, Mo, Cd and Hg. There were no gradients in concentrations of dissolved or solid-phase elements with increasing soil depth. This phenomenon was attributed to physical and biological processes, including the presence and activities of roots and tidal recharge of soil water. Fine dead roots were storage sites for most mineral and trace elements, as some elements in roots composed a significant fraction (5%) of the total soil pool. Analysis of S and Fe concentration differences between live and dead roots suggested extensive formation of pyrite associated with dead roots; correlation analysis suggested that trace metals coprecipitated with pyrite. An analysis of inventories and release/uptake rates indicate turnover of the N, P, Na and Ca soil pools equivalent to other tropical forests; turnover was slow (decades to centuries) for S, Fe, K and trace elements. Our results indicate that mineral and trace element cycling in these soils are characterized by net storage, with net accumulation of most elements much greater than uptake and release by tree roots.     

Pain and compassion in the neonatal unit -- a neonatologist's view

Research has shown that even extremely premature babies are sufficiently developed, anatomically and physiologically, to be capable of experiencing and responding to pain. All newborn infants and especially those who require intensive care in the first days of life are exposed to some painful procedures. Part of the neonatologist's role is the detection and management of pain in these infants. Difficult challenges come with this role. All medications carry known or potential adverse effects and limited research has been done in this vulnerable population. The benefits and risks of all available pain-relieving measures should be balanced when planning management. Compassion is no excuse for a high incidence of undesirable or dangerous side effects. We must proceed with great care.     

SeqExpress: desktop analysis and visualization tool for gene expression experiments

SUMMARY: SeqExpress is a stand-alone desktop application for the identification of relevant genes within collections of microarray or SAGE experiments. A number of analysis, filtering and visualization tools are provided to aid in the selection of groups of genes. If R is installed then the application can use this to provide further analysis. AVAILABILITY: SeqExpress is available at: http://www.seqexpress.com