Lattices of Type I Collagen Select Invasive Variants of K-ras Oncogene-Transfected NIH3T3 with Less Cellular fibronectin

A clone of NIH3T3 transformant (H3) can yield subcutaneous tumors and experimental pulmonary metastasis in nude mice. Compared to H3 in culture, the cells after in vivo tumor growth (H3-N) acquired enhanced tumorigenicity and metastatic ability. Also, indirect immunofluorescence revealed that cellular fibronectin (c-FN) of H3-N was decreased remarkably. We have studied the interactions between H3 and extracellular matrices to elucidate these phenomena. In the present study, we observed the effect of NIH3T3, H3, and H3-N cultured in type I collagen gel. Morphologically in the collagen gel, NIH3T3 assumed an extensive elongated fiber-like shape, H3 assumed a moderately elongated shape, and H3-N assumed a round or spindle shape with short pseudopodia. Compared to conventional cultures on dishes, cell proliferation of all three types was suppressed in collagen gel, but the degree of the suppression was least in H3-N. As a result, H3-N grew fastest in collagen gel. The variants which acquired growth advantage in the subcutaneum of mice also kept it in collagen gel. H3 cells were cultured in type I collagen gel for 4 weeks, a period comparable to that of tumor formation in nude mice. The cells after this long-term culture (H3-C) acquired enhanced tumorigenicity and metastatic ability nearly equal to that of H3-N. FACS analysis revealed that the c-FN of H3-C had decreased to a value comparable to that of H3-N. This means that type I collagen gel as well as subcutaneous tissues could select variants of H3 with less c-FN through proliferation. Moreover, it is suspected that lattices of type I collagen regulate cell proliferation of fibroblast via c-FN. This revised version was published online in August 2006 with corrections to the Cover Date.     

β-Adrenergic modulation of the Na+−K+ pump in frog skeletal muscles

Adrenaline markedly increased the ouabain-sensitive 22Na+-efflux by stimulating the Na+-K+ pump in frog skeletal muscle. The facilitatory effects of adrenaline had the following properties. The effects of adrenaline on the ouabain-sensitive Na+-efflux were observed at concentrations greater than 0.1 microM and the magnitude increased with concentration up to 10 microM. At a concentration of 30 microM, adrenaline markedly augmented the ouabain-sensitive Na+-efflux, but other biogenic amines were less effective (noradrenaline and dopamine) or ineffective (histamine and serotonin). The increase of Na+-efflux induced by 1 microM adrenaline was blocked by 3 microM propranolol, but not by 3 microM phenoxybenzamine. The properties of the facilitatory action of adrenaline on the ouabain-sensitive Na+-efflux suggest that beta-adrenoceptors have an important role in modulating the Na+-K+ pump activity in the skeletal muscle membrane. The protein complex localized in excitable membranes, namely the Na+-K+ ATPase-beta-adrenoceptor complex, may be the functional unit which operates the membrane machinery driving the Na+-K+ pump.     

Fibrinogen stabilizes placental-maternal attachment during embryonic development in the mouse

In humans, maternal fibrinogen (Fg) is required to support pregnancies by maintaining hemostatic balance and stabilizing uteroplacental attachment at the fibrinoid layer found at the fetal-maternal junction. To examine relationships between low Fg levels and early fetal loss, a genetic model of afibrinogenemia was developed. Pregnant mice homozygous for a deletion of the Fg-gamma chain, which results in a total Fg deficiency state (FG(-/-)), aborted the fetuses at the equivalent gestational stage seen in humans. Results obtained from timed matings of FG(-/-) mice showed that vaginal bleeding was initiated as early as embryonic day (E)6 to 7, a critical stage for maternal-fetal vascular development. The condition of afibrinogenemia retarded embryo-placental development, and consistently led to abortion and maternal death at E9.75. Lack of Fg did not alter the extent or distribution pattern of other putative factors of embryo-placental attachment, including laminin, fibronectin, and Factor XIII, indicating that the presence of fibrin(ogen) is required to confer sufficient stability at the placental-decidual interface. The results of these studies demonstrate that maternal Fg plays a critical role in maintenance of pregnancy in mice, both by supporting proper development of fetal-maternal vascular communication and stabilization of embryo implantation.     

The effect of available choices on ambiguity aversion: an examination of different types of ambiguity

Ellsberg's two-color problem, known as an example of ambiguity aversion, has generated a great deal of interest among many researchers. However, an unsettled question remains regarding the conditions that lead to heightened or diminished ambiguity aversion. We conducted a series of experiments, which required participants to choose between a clear and vague bet. Results showed that participants did not always prefer the clear bet, and the ratios of those who indicated ambiguity aversion varied depending on the types of ambiguity. Furthermore, ambiguity aversion consistently decreased when participants were allowed to choose the ambiguity task they would perform. These results were interpreted in terms of the pattern of second order probability distributions and illusion of control.     

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Selective loss of gastrointestinal mast cells and impaired immunity in PI3K-deficient mice

Mice that lack the p85alpha regulatory subunit of phosphatidylinositol-3 kinase (PI3K) are deficient in gastrointestinal and peritoneal mast cells but have dermal mast cells. Accordingly, these mice show impaired bacterial clearance in response to acute septic peritonitis and are highly susceptible to infection by the intestinal nematode Strongyloides venezuelensis. Systemic anaphylactic shock responses, however, are intact. We found that although reconstitution of PI3Kminus sign/minus sign mice with bone marrow--derived mast cells (BMMCs) restored anti-bacterial immunity, only T helper type 2 (TH2)-conditioned BMMCs, not "standard" BMMCs, were able to restore anti-nematode immunity. This finding highlights the importance of the TH2 response in the control of nematode infection. Thus, PI3K likely plays an essential role in host immune responses by regulating both the development and induction of mast cells.     

In vivo labeling of amyloid with BF-108

Detection of aggregated amyloid-beta (Abeta) with a non-invasive imaging modality such as positron emission tomography (PET) was suggested to be ideal for the diagnosis of Alzheimer's disease (AD) prior to the onset of clinical symptoms. We have been searching for imaging probe candidates with a high affinity for aggregated Abeta in vitro and in vivo and high lipophilicity, a characteristic that allows for the permeation of the blood-brain barrier (BBB). As analyzed by Thioflavin T (ThT) assay and octanol/water partition coefficient test (PC), 3-diethylamino-6-(2-fluoroethyl)ethylaminoacridine (BF-108) were found to have high affinity for Abeta aggregates in vitro and high lipophilicity. Intravenously administrated BF-108 labeled Abeta aggregates injected into the amygdala as observed under a fluorescence microscope, showing this compound's permeability of BBB and an ability to label Abeta in vivo. BF-108 also labeled neuritic senile plaques (SPs), neurofibrillary tangles, and amyloid-laden vessels in temporal and hippocampal sections from AD patients. Following intravenous administration of BF-108 to an APP23 transgenic (TG) mouse, in vivo labeling of endogenous plaques was seen in brain sections by fluorescence microscopy. These properties suggest the potential utility of BF-108 for in vivo imaging of AD pathology.     

Pseudotype hepatitis C virus enters immature myeloid dendritic cells through the interaction with lectin

Dendritic cells (DC) are the most potent antigen-presenting cells that regulate immune responses. One of the mechanisms for hepatitis C virus (HCV) persistence is the ability of HCV to suppress DC function. Direct HCV infection to blood DC has been implicated for DC dysfunction. To clarify the susceptibility of each DC subset to HCV, we used pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins (E1 and E2). We demonstrate that pseudotype VSV enters myeloid DC (MDC) but not plasmacytoid DC (PDC). The highest efficiency of pseudotype VSV entry to MDC was observed when MDC were cultured with GM-CSF. Such efficiency decreased when MDC are matured with the treatment of IL-4, CpG oligodeoxynucleotide, or CD40 ligand. Mannan inhibited pseudotype VSV entry to MDC, but Ca(2+) chelators failed to do so. These results show that pseudotype VSV possessing HCV-E1 and E2 enters immature MDC through the interaction with lectins in a Ca(2+)-independent manner.