Dancing the tight rope on the nanoscale--Calibrating a heat flux sensor of a scanning thermal microscope.

We report on a precise in situ procedure to calibrate the heat flux sensor of a near-field scanning thermal microscope. This sensitive thermal measurement is based on 1ω modulation technique and utilizes a hot wire method to build an accessible and controllable heat reservoir. This reservoir is coupled thermally by near-field interactions to our probe. Thus, the sensor's conversion relation V(th)(Q(GS)*) can be precisely determined. V(th) is the thermopower generated in the sensor's coaxial thermocouple and Q(GS)* is the thermal flux from reservoir through the sensor. We analyze our method with Gaussian error calculus with an error estimate on all involved quantities. The overall relative uncertainty of the calibration procedure is evaluated to be about 8% for the measured conversion constant, i.e., (2.40 ± 0.19) µV/µW. Furthermore, we determine the sensor's thermal resistance to be about 0.21 K/µW and find the thermal resistance of the near-field mediated coupling at a distance between calibration standard and sensor of about 250 pm to be 53 K/µW.

Microencapsulation technology by nature: Cell derived extracellular vesicles with therapeutic potential.

Cell derived extracellular vesicles are submicron structures surrounded by phospholipid bilayer and released by both prokaryotic and eukaryotic cells. The sizes of these vesicles roughly fall into the size ranges of microbes, and they represent efficient delivery platforms targeting complex molecular information to professional antigen presenting cells. Critical roles of these naturally formulated units of information have been described in many physiological and pathological processes. Extracellular vesicles are not only potential biomarkers and possible pathogenic factors in numerous diseases, but they are also considered as emerging therapeutic targets and therapeutic vehicles. Strikingly, current drug delivery systems, designed to convey therapeutic proteins and peptides (such as liposomes), show many similarities to extracellular vesicles. Here we review some aspects of therapeutic implementation of natural, cell-derived extracellular vesicles in human diseases. Exploration of molecular and functional details of extracellular vesicle release and action may provide important lessons for the design of future drug delivery systems.

Compact device for cleaning scanner-mounted scanning tunneling microscope tips using electron bombardment.

Most scanning probe techniques rely on the assumption that both sample and tip are free from adsorbates, residues, and oxide not deposited intentionally. Getting a clean sample surface can be readily accomplished by applying ion sputtering and subsequent annealing, whereas finding an adequate treatment for tips is much more complicated. The method of choice would effectively desorb undesired compounds without reducing the sharpness or the general geometry of the tip. Several devices which employ accelerated electrons to achieve this are described in the literature. To minimize both the effort to implement this technique in a UHV chamber and the overall duration of the cleaning procedure, we constructed a compact electron source fitted into a sample holder, which can be operated in a standard Omicron variable-temperature (VT)-STM while the tip stays in place. This way a maximum of compatibility with existing systems is achieved and short turnaround times are possible for tip cleaning.

T lymphocytes are targets for platelet- and trophoblast-derived microvesicles during pregnancy.

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)-PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV-lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

Magnetism in SQUIDs at millikelvin temperatures.

We have characterized the temperature dependence of the flux threading dc SQUIDs cooled to millikelvin temperatures. The flux increases as 1/T as temperature is lowered; moreover, the flux change is proportional to the density of trapped vortices. The data are compatible with the thermal polarization of surface spins in the trapped fields of the vortices. In the absence of trapped flux, we observe evidence of spin-glass freezing at low temperature. These results suggest an explanation for the universal 1/f flux noise in SQUIDs and superconducting qubits.

The role of N-methyl-D-aspartate receptors and nitric oxide in cochlear dopamine release.

Dopamine (DA) released from lateral olivocochlear (LOC) terminals may have a neuroprotective effect in the cochlea. To explore the role of N-methyl-d-aspartate (NMDA) receptors and nitric oxide (NO) in the modulation of a cochlear DA release, we measured the release of [3H]DA from isolated mouse cochlea in response to the application of NMDA. NMDA at 100 muM significantly increased the electrical-field stimulation-evoked and resting release of DA from the cochlea. The NO donor sodium nitroprusside enhanced the basal outflow of DA but failed to influence the evoked release. The administration of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) alone was ineffective, but it significantly inhibited the initial phase of the NMDA-induced elevation of DA outflow, which suggested the role of NO in the NMDA-induced DA release. The DA uptake inhibitor nomifensine increased the electrically evoked release of DA. Nomifensine failed to change the effect of NMDA on the resting or electrically-evoked DA release, which suggested that the uptake mechanism does not play a role in NMDA-evoked and NO-mediated DA release. In summary, we provide evidence that NO can modulate the release of DA from the cochlea following NMDA receptor activation, but does not affect the uptake of DA.

D2 autoreceptor inhibition reveals oxygen-glucose deprivation-induced release of dopamine in guinea-pig cochlea.

Dopamine (DA), released from the lateral olivocochlear (LOC) efferent terminals, the efferent arm of the short-loop feedback in the cochlea, is considered as a protective factor in the inner ear since it inhibits auditory nerve dendrite firing in ischemia- or noise-induced excitotoxicity leading to sensorineural hearing loss (SNHL). In the present study we investigated the effect of oxygen-glucose deprivation (OGD), an in vitro ischemia model, on guinea-pig cochlear [(3)H]DA release in a microvolume superfusion system. We found that OGD alone failed to induce a detectable elevation of [(3)H]DA level, but in the presence of specific D(2) receptor antagonists, sulpiride and L-741,626, it evoked a significant increase in the extracellular concentration of [(3)H]DA. D(2) negative feedback receptors are involved not exclusively in the regulation of synthesis and vesicular release of DA, but also in the activation of its reuptake. Thus, D(2) receptor antagonism interferes with the powerful reuptake of DA from the extracellular space. To explore the underlying mechanism of this DA-releasing effect we applied nomifensine and found that the effect of OGD on cochlear DA release in the presence of D(2) antagonists could be inhibited by this selective DA uptake inhibitor. This finding indicates that the OGD-evoked DA release was mainly mediated through the reverse operation of the DA transporter. The two structurally different D(2) antagonists also augmented the electrical field stimulation-evoked release of DA proving the presence of D(2) autoreceptors on dopaminergic LOC terminals. Our results confirm the presence and role of D(2) DA autoreceptors in the regulation of DA release from LOC efferents, and suggest a protective local mechanism during ischemia which involves the direct transporter-mediated release of DA. Increasing the release of the protective transmitter DA locally in the inner ear may form the basis of future new therapeutic strategies in patients suffering from SNHL.

Co-localization of P2Y1 receptor and NTPDase1/CD39 within caveolae in human placenta.

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) is the dominant ecto-nucleotidase of vascular and placental trophoblastic tissues and appears to modulate the functional expression of type-2 purinergic (P2) G-protein coupled receptors (GPCRs). Hence, this ectoenzyme could regulate nucleotide-mediated signalling events in placental tissue. This immunohistochemical and immuno-electron microscopic study demonstrates the expression of NTPDase1/CD39, P2Y1 and P2Y2 receptors in different cell types of human placenta. Specifically P2Y1 has an exclusive vascular distribution whereas P2Y2 is localized on trophoblastic villi. Co-localization of P2Y1 and NTPDase1/CD39 are observed in caveolae, membrane microdomains of endothelial cells. The differential localization of these P2 receptors might indicate their unique roles in the regulation of extracellular nucleotide concentrations in human placental tissues and consequent effects on vascular tone and blood fluidity.

Extracellular calcium sensing receptor in human pancreatic cells.

BACKGROUND AND AIMS: The extracellular calcium sensing receptor (CaR) plays a key role in the calcium homeostatic system and is therefore widely expressed in tissues involved in calcium metabolism. However, the CaR has also been identified in other tissues where its role is less clear. We have investigated the presence of the CaR in the human pancreas. METHODS: Messenger RNA for the CaR was detected by reverse transcription-polymerase chain reaction and the protein was localised by immunostaining. CaR function was assayed in Capan-1 cells by measuring intracellular calcium and [(3)H] thymidine incorporation. RESULTS: The receptor was highly expressed in human pancreatic ducts. It was also expressed in exocrine acinar cells, in islets of Langerhans, and in intrapancreatic nerves and blood vessels. The CaR was expressed in both normal and neoplastic human tissue samples but was detected in only one of five ductal adenocarcinoma cells lines examined. Experiments on the CaR expressing adenocarcinoma cell line Capan-1 showed that the CaR was functional and was linked to mobilisation of intracellular calcium. Stimulation of the CaR reduced Capan-1 cell proliferation. CONCLUSIONS: We propose that the CaR may play multiple functional roles in the human pancreas. In particular, the CaR on the duct luminal membrane may monitor and regulate the Ca(2+) concentration in pancreatic juice by triggering ductal electrolyte and fluid secretion. This could help to prevent precipitation of calcium salts in the duct lumen. The CaR may also help to regulate the proliferation of pancreatic ductal cells.

Dynamics and control of a multimode laser: Reduction of space-dependent rate equations to a low-dimensional system.

We suggest a quantitatively correct procedure for reducing the spatial degrees of freedom of the space-dependent rate equations of a multimode laser that describe the dynamics of the population inversion of the active medium and the mode intensities of the standing waves in the laser cavity. The key idea of that reduction is to take advantage of the small value of the parameter that defines the ratio between the population inversion decay rate and the cavity decay rate. We generalize the reduction procedure for the case of an intracavity frequency doubled laser. Frequency conversion performed by an optically nonlinear crystal placed inside the laser cavity may cause a pronounced instability in the laser performance, leading to chaotic oscillations of the output intensity. Based on the reduced equations, we analyze the dynamical properties of the system as well as the problem of stabilizing the steady state. The numerical analysis is performed considering the specific system of a Nd:YAG (neodymium-doped yttrium aluminum garnet) laser with an intracavity KTP (potassium titanyl phosphate) crystal.

Cultured astrocytes react to LPS with increased cyclooxygenase activity and phagocytosis.

Phagocytosis and prostaglandin E(2) production were investigated in purified cultures of perinatal rat forebrain astrocytes. Light and electron microscopic data indicated that astrocytes respond to bacterial endotoxin, lipopolysaccharide (LPS) by increased phagocytosis and by activating the cyclooxygenase enzyme-pathway. LPS-inducible phagocytosis of astrocytes was demonstrated by electron microscopic studies on colloidal gold uptake and by photometric determination of fluorescent bead ingestion. The internalisation of fragments of the plasma membrane was shown by histochemical detection of membrane-bound ecto-ATPase activity within intracellular vesicles. Activation of the cyclooxygenase pathway, a characteristic reaction of immune cells under inflammatory conditions, was also detected in astroglial cells upon treatment with LPS. The increased prostaglandin E(2) (PGE(2)) production by astrocytes in response to LPS was reduced by the non-steroid anti-inflammatory drug, indomethacin. Our data indicate that astrocytes display some tissue-protective reactions in response to inflammation inducing factors, even in the absence of peripheral immune cells or central microglia. The role of inducible astrocytic phagocytosis in a non-immune protection-pathway is discussed.

Stabilization of an unstable steady state in intracavity frequency-doubled lasers

We predict theoretically that it is possible to stabilize the steady state in multimode, intracavity doubled, diode pumped Nd:YAG (neodymium-doped yttrium aluminum garnet) lasers using two output signals, namely, the sum intensities of the infrared laser modes polarized in two different orthogonal directions (X and Y) and one feedback input parameter, the pump rate. The stabilization is possible for arbitrarily large numbers of modes polarized in the X and Y direction. Different strategies of stabilization based on proportional feedback, derivative control, and their combination are discussed. The analytical and numerical results of the linear control theory are illustrated with numerical simulations of the underlying nonlinear differential equations. We show that one can maintain the stable steady state of the laser output for an arbitrarily large pump rate by taking advantage of a tracking procedure.

Home rehabilitation for older adults with fractured hips: how many will take part?

Rehabilitation at home is a new 'technology' which has been promoted as an efficient alternative to hospital rehabilitation for older patients with conditions such as fractured hip. In Australia, no formal description of elderly patients with fractured hips likely to be eligible for home rehabilitation has been made and the acceptability of such services is unclear. Using information obtained prospectively from a consecutive sample of 188 patients with a fractured hip we describe the characteristics of older adults who were eligible for a trial examining home versus hospital rehabilitation. While staff assessed 36% of patients as eligible, only 20% were both eligible and agreeable. Reasons for refusal to participate included a preference for inpatient rehabilitation (26%), family reluctance (26%) and anxiety regarding the ability to manage at home (16%). Our results suggest that home rehabilitation is suitable for the least disabled group but is still unacceptable to many elderly patients and their families. As the population ages and hip fractures increase, home rehabilitation in its current form will have little impact on future bed needs.

Simulation of multilane freeway traffic with detailed rules deduced from microscopic driving behavior

A simulation to model traffic on a multilane freeway is introduced starting from microscopic driving rules. The model takes each individual car into account with its individual features and actual situations, so that a distribution of parameters as well as different behaviors can easily be analyzed. Therefore, a detailed study of certain situations, driving tactics, vehicle properties, and their influence on the global traffic flow can be performed. The model is discussed, as are first results such as the influence of driver behavior on the fundamental diagram and, in addition, the dynamics of microscopic, individual quantities like separation and difference in speed between successive cars. It turns out that a hysteresis in the reaction of the driver for speeding up and slowing down plays an important role, and effects macroscopic quantities like the shape of the fundamental diagram, e.g., the metastable behavior around the maximum flow and on the speed of observed jams running backward. Furthermore, microscopic time resolved characteristics are strongly influenced, e.g., oscillations in the distance and relative speed between successive cars.

Purification, characterization, and localization of an ATP diphosphohydrolase in porcine kidney.

Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.

Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1.

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.

Palmitoylation targets CD39/endothelial ATP diphosphohydrolase to caveolae.

Ectonucleotidases influence purinergic receptor function by the hydrolysis of extracellular nucleotides. CD39 is an integral membrane protein that is a prototype member of the nucleoside 5'-triphosphate diphosphohydrolase family. The native CD39 protein has two intracytoplasmic and two transmembrane domains. There is a large extracellular domain that undergoes extensive glycosylation and can be post-translationally modified by limited proteolysis. We have identified a potential thioester linkage site for S-acylation within the N-terminal region of CD39 and demonstrate that this region undergoes palmitoylation in a constitutive manner. The covalent lipid modification of this region of the protein appears to be important both in plasma membrane association and in targeting CD39 to caveolae. These specialized plasmalemmal domains are enriched in G protein-coupled receptors and appear to integrate cellular activation events. We suggest that palmitoylation could modulate the function of CD39 in regulating cellular signal transduction pathways.

CD39 as a caveolar-associated ectonucleotidase.

CD39 is a human lymphoid cell activation antigen, (also referred to E-ATPDase or apyrase) that hydrolyzes extracellular ATP and ADP. Although it has been widely studied, its physiological role, however, still remains unclear. This ectonucleotidase generally is said to be evenly distributed in the membrane of the cells. However, we observed that in cell types which possess caveolae, specialised membrane invaginations involved in signalling, CD39 is preferentially targeted to these membrane microdomains. Since all molecules involved in signalling (eNOS, G-proteins, receptors) which are targeted to the caveolae undergo posttranslational modifications (e.g., palmitoylation) we hypothesize the same to be the case for CD39. Furthermore, its presence in the caveolae supports its participation in signalling events.

Ultrastructural localization of beta-arrestin-1 and -2 in rat lumbar spinal cord.

beta-arrestins play significant roles in agonist-mediated desensitization of G protein-coupled receptors. Although the presence of beta-arrestin subtypes, beta-arrestin-1 and(- 2) in rat brain has been studied extensively, their existence in the spinal cord has not been described. In the current study, we performed immunohistochemical analyses of beta-arrestins at both light and electron microscopic levels using rat lumbar 1-2 spinal cord segments. Intense immunoreactivity for beta-arrestin-1 was found in the motoneurons in lamina IX of the ventral horn and elongated cells in the dorsal nucleus of Clarke. Modest immunoreactivity was detected among the neurons of laminae V and VII/VIII, and weaker immunoreactivity in laminae III, IV, and X. beta-arrestin-2 immunoreactivity was also distributed through laminae III-X in the order of IX > dorsal nucleus of Clarke > V > VII/VIII > IV > III > X. Laminae I and II did not show immunoreactivity. At the electron microscopic level, both beta-arrestin-immunoreactive and nonimmunoreactive dendrites were observed, whereas axons and terminal boutons were devoid of immunoreactivity. In immunoreactive dendrites most beta-arrestin immunoreactivity was distributed throughout the cytoplasm, demonstrating their association with microtubules. In addition, strong immunoreactivity was often found at postsynaptic densities. Our results thus suggest beta-arrestins' possible involvement in both motor and sensory mechanisms at the postsynaptic level in rat lumbar spinal cord.