Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.     

Three-dimensional registration of histology of human atherosclerotic carotid plaques to in-vivo imaging

An accurate spatial relationship between 3D in-vivo carotid plaque and lumen imaging and histological cross sections is required to study the relationship between biomechanical parameters and atherosclerotic plaque components. We present and evaluate a fully three-dimensional approach for this registration problem, which accounts for deformations that occur during the processing of the specimens. By using additional imaging steps during tissue processing and semi-automated non-linear registration techniques, a 3D-reconstruction of the histology is obtained.The methodology was evaluated on five specimens obtained from patients, operated for severe atherosclerosis in the carotid bifurcation. In more than 80% of the histology slices, the quality of the semi-automated registration with computed tomography angiography (CTA) was equal to or better than the manual registration. The inter-observer variability was between one and two in-vivo CT voxels and was equal to the manual inter-observer variability. Our technique showed that the angles between the normals of the registered histology slices and the in-vivo CTA scan direction ranged 6–56°, indicating that proper 3D-registration is crucial for establishing a correct spatial relation with in-vivo imaging modalities. This new 3D-reconstruction technique of atherosclerotic plaque tissue opens new avenues in the field of biomechanics as well as in the field of image processing, where it can be used for validation purposes of segmentation algorithms.     

MRI-based quantification of outflow boundary conditions for computational fluid dynamics of stenosed human carotid arteries

Accurate assessment of wall shear stress (WSS) is vital for studies on the pathogenesis of atherosclerosis. WSS distributions can be obtained by computational fluid dynamics (CFD) using patient-specific geometries and flow measurements. If patient-specific flow measurements are unavailable, in- and outflow have to be estimated, for instance by using Murray’s Law. It is currently unknown to what extent this law holds for carotid bifurcations, especially in cases where stenoses are involved. We performed flow measurements in the carotid bifurcation using phase-contrast MRI in patients with varying degrees of stenosis. An empirical relation between outflow and degree of area stenosis was determined and the outflow measurements were compared to estimations based on Murray’s Law. Furthermore, the influence of outflow conditions on the WSS distribution was studied.For bifurcations with an area stenosis smaller than 65%, the outflow ratio of the internal carotid artery (ICA) to the common carotid artery (CCA) was 0.62±0.12 while the outflow ratio of the external carotid artery (ECA) was 0.35±0.13. If the area stenosis was larger than 65%, the flow to the ICA decreased linearly to zero at 100% area stenosis. The empirical relation fitted the flow data well (R²=0.69), whereas Murray’s Law overestimated the flow to the ICA substantially for larger stenosis, resulting in an overestimation of the WSS. If patient-specific flow measurements of the carotid bifurcation are unavailable, estimation of the outflow ratio by the presented empirical relation will result in a good approximation of calculated WSS using CFD.     

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.     

EphrinA1 repulsive response is regulated by an EphA2 tyrosine phosphatase

Ephrin kinases and their ephrin ligands transduce repulsion of cells in axon guidance, migration, invasiveness, and tumor growth, exerting a negative signaling on cell proliferation and adhesion. A key role of their kinase activity has been confirmed by mutant kinase inactive receptors that shift the cellular response from repulsion to adhesion. Our present study aimed to investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in ephrinA1/EphA2 signaling. LMW-PTP, by means of dephosphorylation of EphA2 kinase, negatively regulates the ephrinA1-mediated repulsive response, cell proliferation, cell adhesion and spreading, and the formation of retraction fibers, thereby confirming the relevance of the net level of tyrosine phosphorylation of Eph receptors. LMW-PTP interferes with ephrin-mediated mitogen-activated protein kinase signaling likely through inhibition of p120RasGAP binding to the activated EphA2 kinase, thereby confirming the key role of mitogen-activated protein kinase inhibition by ephrinA1 repulsive signaling. We conclude that LMW-PTP acts as a terminator of EphA2 signaling causing an efficient negative feedback loop on the biological response mediated by ephrinA1 and pointing on tyrosine phosphorylation as the main event orchestrating the repulsive response.     

HLA antigen frequency in the Koya tribe of Andhra Pradesh, India

The frequencies of HLA-A, -B, and -C antigens were studied in a tribal population of Koya from Andhra Pradesh in southern India. No other well-defined tribal population has been studied with which the present results may be compared. However, the HLA profile of Koya showed distinct differences from the general HLA distribution in India in the frequency of a large number of antigens both at the A and B loci. This study indicates the distinctiveness of this tribal population and suggests the potential importance of the study of HLA frequencies in tribal groups of India.     

The influence of boundary conditions on wall shear stress distribution in patients specific coronary trees

Patient specific geometrical data on human coronary arteries can be reliably obtained multislice computer tomography (MSCT) imaging. MSCT cannot provide hemodynamic variables, and the outflow through the side branches must be estimated. The impact of two different models to determine flow through the side branches on the wall shear stress (WSS) distribution in patient specific geometries is evaluated. Murray's law predicts that the flow ratio through the side branches scales with the ratio of the diameter of the side branches to the third power. The empirical model is based on flow measurements performed by Doriot et al. (2000) in angiographically normal coronary arteries. The fit based on these measurements showed that the flow ratio through the side branches can best be described with a power of 2.27. The experimental data imply that Murray's law underestimates the flow through the side branches. We applied the two models to study the WSS distribution in 6 coronary artery trees. Under steady flow conditions, the average WSS between the side branches differed significantly for the two models: the average WSS was 8% higher for Murray's law and the relative difference ranged from -5% to +27%. These differences scale with the difference in flow rate. Near the bifurcations, the differences in WSS were more pronounced: the size of the low WSS regions was significantly larger when applying the empirical model (13%), ranging from -12% to +68%. Predicting outflow based on Murray's law underestimates the flow through the side branches. Especially near side branches, the regions where atherosclerotic plaques preferentially develop, the differences are significant and application of Murray's law underestimates the size of the low WSS region.     

Astrocytes, spontaneity, and the developing thalamus.

Recent studies in the ventrobasal (VB) thalamus have shown that astrocytes display spontaneous intracellular calcium [Ca(2+)](i) oscillations early postnatally. [Ca(2+)](i) oscillations are correlated in groups of up to five astrocytes, and propagate between cells. NMDA receptor-mediated, long lasting inward currents in thalamocortical (TC) neurons of the VB complex are correlated to [Ca(2+)](i) increases in neighbouring astrocytes, and stimulation of astrocytic [Ca(2+)](i) increases also lead to inward currents in neurons. These findings suggest that astrocytes are spontaneously active and can induce neuronal activity, a reversal of the previously held view of neuron-glia interactions in the central nervous system. This activity occurs at an important period in the development of the thalamus and therefore suggests a potential functional role in a variety of processes. Along with data on the neurotransmitter receptor repertoire of thalamic astrocytes these findings enlarge the body of knowledge on astrocytes in the thalamus, and further contribute to the emerging field of astrocyte-neuron and neuron-astrocyte interactions in the central nervous system.