Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods.

Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes.

Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics.

Background: Bloodstream infections (BSI) remain a major challenge with high mortality rate, with an incidence that is increasing worldwide. Early treatment with appropriate therapy can reduce BSI-related morbidity and mortality. However, despite recent progress in molecular based assays, complex sample preparation steps have become critical roadblock for a greater expansion of molecular assays.

Microfluidic-Based Bacteria Isolation from Whole Blood for Diagnostics of Blood Stream Infection.

Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods.

Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics.

Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood.

Genotyping of single nucleotide polymorphisms by melting curve analysis using thin film semi-transparent heaters integrated in a lab-on-foil system.

The recent technological advances in micro/nanotechnology present new opportunities to combine microfluidics with microarray technology for the development of small, sensitive, single-use, point-of-care molecular diagnostic devices. As such, the integration of microarray and plastic microfluidic systems is an attractive low-cost alternative to glass based microarray systems. This paper presents the integration of a DNA microarray and an all-polymer microfluidic foil system with integrated thin film heaters, which demonstrate DNA analysis based on melting curve analysis (MCA).

Binding of losartan to angiotensin AT1 receptors increases dopamine D1 receptor activation.

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro.

Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis.

Loss of water through the immature skin can lead to hypothermia and dehydration in preterm infants. The water and glycerol channel aquaglyceroporin-3 (AQP3) is abundant in fetal epidermis and might influence epidermal water handling and transepidermal water flux around birth. To investigate the role of AQP3 in immature skin, we measured in vivo transepidermal water transport and AQP3 expression in rat pups exposed to clinically relevant fluid homeostasis perturbations.

Ouabain protects against adverse developmental programming of the kidney.

The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards the formation of functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease.

Analysis of mice with targeted deletion of AQP9 gene provides conclusive evidence for expression of AQP9 in neurons.

AQP9 is an aquaglyceroporin that serves important functions in peripheral organs, including the liver. Reflecting the lack of AQP9 knockout mice, uncertainties still prevail regarding the localization and roles of AQP9 in the central nervous system. Here we present a comprehensive analysis of AQP9 gene expression in brain, based on a quantitative and multipronged approach that includes the use of animals with targeted deletion of the AQP9 gene.

Negative reciprocity between angiotensin II type 1 and dopamine D1 receptors in rat renal proximal tubule cells.

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling.

Identification of a molecular target for glutamate regulation of astrocyte water permeability.

Astrocytes play a key role for maintenance of brain water homeostasis, but little is known about mechanisms of short-term regulation of astrocyte water permeability. Here, we report that glutamate increases astrocyte water permeability and that the molecular target for this effect is the aquaporin-4 (AQP4) serine 111 residue, which is in a strategic position for control of the water channel gating. The glutamate effect involves activation of group I metabotropic glutamate receptors (mGluR), intracellular calcium release, and activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and nitric oxide synthase (NOS).

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells.

Intracellular dynamics of calcyon, a neuron-specific vesicular protein.

Calcyon is a brain-specific protein, implicated in clathrin-mediated endocytosis. In this descriptive study we show that calcyon is exclusively expressed in neurons, and localized in moving vesicles. The movement of calcyon-containing vesicles was dependent on temperature and on intact microtubules, in addition these vesicles were colocalized with a marker for endocytosed plasma membrane proteins, suggesting that calcyon vesicles follow the endocytic recycling pathway.

Distribution of aquaporin 4 on sarcolemma of fast-twitch skeletal myofibres.

The aquaporin 4 (AQP4) water channel is present on the sarcolemma of fast-twitch-type skeletal myofibres. We have examined the distribution of AQP4 in relation to sarcolemmal domain structure and found that AQP4 protein is not evenly distributed on the sarcolemma. Immunofluorescence staining of isolated single myofibres indicated a punctate staining pattern overlapping with the dystrophin glycoprotein complex, but with the transverse tubule openings being left clear.

Effect of TNF-alpha on CD3-zeta and MHC-I in postnatal rat hippocampus.

The zeta subunit of the CD3 T-cell receptor complex and the major histocompatibility complex class 1 (MHC-I) are important not only for the immune response to antigens, they also function as signal molecules in the brain, where they play a role in the postnatal maturation process. The expression of these molecules can be regulated by cytokines. In situations associated with increased cytokine production, such as neonatal hypoxia, the hippocampus is particularly susceptible to permanent damage.

Low doses of ouabain protect from serum deprivation-triggered apoptosis and stimulate kidney cell proliferation via activation of NF-kappaB.

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP3R) triggers slow calcium oscillations and activation of NF-kappaB. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth.

Allosteric changes of the NMDA receptor trap diffusible dopamine 1 receptors in spines.

The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors.

Brain mitochondria contain aquaporin water channels: evidence for the expression of a short AQP9 isoform in the inner mitochondrial membrane.

Aquaporins are a family of water channels found in animals, plants, and microorganisms. A subfamily of aquaporins, the aquaglyceroporins, are permeable for water as well as certain solutes such as glycerol, lactate, and urea. Here we show that the brain contains two isoforms of AQP9--an aquaglyceroporin with a particularly broad substrate specificity--and that the more prevalent of these isoforms is expressed in brain mitochondria.

Water channels (aquaporins) and their role for postnatal adaptation.

Birth is a transition from an underwater life in the uterus to a terrestrial life in a milieu where supply of water is limited. Rapid adaptation to the new environment is crucial for survival and health of infants. The discovery of a family of molecules-aquaporin (AQP) water channels-that are responsible for regulated water transport across cell membranes has made it possible to identify the molecular mechanisms behind the postnatal homeostatic adaptation and to better understand water imbalance-related disorders in infancy and childhood.

Copper inhibits the water and glycerol permeability of aquaporin-3.

Aquaporin-3 (AQP3) is an aquaglyceroporin expressed in erythrocytes and several other tissues. Erythrocytes are, together with kidney and liver, the main targets for copper toxicity. Here we report that both water and glycerol permeability of human AQP3 is inhibited by copper.

Cellular response to renal hypoxia is different in adolescent and infant rats.

Immature renal tubules are more tolerant to ischemia than mature renal tubules. Here we compared the developmental pattern for some cellular responses evoked by hypoxia and reoxygenation in renal proximal tubules from 10- and 40-day-old rats. Redistribution of Na(+)-K(+)-ATPase from the plasma membrane was studied by confocal microscopy techniques in primary cultured renal proximal tubular cells.

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations.

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells.

Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells.

Nickel is a common cause of pneumoconiosis. Here, we show that nickel inactivates aquaporin (AQP)-3, the water channel expressed apically in epithelial cells of human terminal airways. Human AQP3 was transiently transfected into human lung cells, and water permeability was measured in transfected and neighboring untransfected cells.

Transepidermal water loss in developing rats: role of aquaporins in the immature skin.

In the extremely preterm infant, high transepidermal water loss (TEWL) can result in severe dehydration. TEWL has been attributed to the structural properties of the epidermis but might also be influenced by mechanisms that facilitate water transport. To investigate whether aquaporins (AQP) may be involved in the extreme losses of water through immature skin, we examined the presence and cellular distributions of AQP-1 and AQP-3 in embryonic and adult rat skin by immunohistochemistry.

Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine.

Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (P(f)) was measured in individual LLC-PK1 cells that were transiently transfected with AQP4.