Delayed separation and the plasma amino acids arginine and ornithine

Induction of gap junctional communication (GJC) is discussed as one possible mechanism underlying the cancer-preventive properties of carotenoids. Structurally different carotenoids have different effects on this GJC pathway. Beta-carotene, echinenone, canthaxanthin, cryptoxanthin and 4-hydroxy-beta-carotene efficiently induce GJC in murine fibroblasts. Decomposition products of canthaxanthin (e.g. 4-oxo-retinoic acid) enhance both GJC and expression of connexin 43 mRNA. Thus, the biological activity of canthaxanthin is due at least in part to formation of active decomposition products such as 4-oxo-retinoic acid. A number of other small molecules such as 1,25-dihydroxycholecalciferol or thyroid hormones serve as nuclear receptor ligands. Cholecalciferol, 3,3',5-triiodo-L-thyronine and L-thyroxine induce GJC to a similar extent as carotenoids or retinoic acid. Interactions between these signalling pathways may be involved in GJC regulation.     

In vitro studies of cochlear excitation

Cells in tissues coordinate their activity by sharing ions, second messengers, and small metabolites through clusters of intercellular channels called gap junctions. The thyroid hormones 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) are capable of modulating gap junctional communication (GJC) as are 1,25-dihydroxyvitamin D3, retinoic acid, and other nuclear receptor ligands. T3 and T4 were found to stimulate GJC in WB-F344 rat liver epithelial cells dose-dependently at concentrations between 1 nM and 0.1 microM, assayed by the dye transfer method using Lucifer Yellow CH. The stimulation of cell-cell communication was preceded by an increase in connexin43 mRNA levels and was accompanied by an accumulation of connexin43 protein measurable 2 days after incubation with these compounds. These observations establish a novel role of thyroid hormones in the regulation of gap junctional intercellular communication via connexin43 gene expression.     

Emergence of undifferentiated rat tracheal cell carcinomas, but not squamous cell carcinomas, is associated with a loss of expression of E-cadherin and of gap junction communication

A series of cells representing normal, non-tumorigenic cell lines, as well as differentiating neoplastic and undifferentiated neoplastic rat tracheal epithelial cell populations were evaluated for their ability to establish homologous and/or heterologous cell-cell gap junction communication in culture. Gap junction communication was evaluated by flow cytometric quantitation of the transfer of the fluorescent dye calcein from a donor to a recipient cell population via gap junctions. The data indicate that normal primary cultures of rat tracheal epithelial cells, as well as non-tumorigenic cell lines and squamous cell carcinomas cell populations, retain the ability to establish both homologous and heterologous gap junction communication. In all cases an average of >48% of recipient cells had acquired calcein label during a 5-h interval of co-culture of donor and recipient cells at confluent densities. Cells harvested directly from squamous cell carcinoma tumors exhibited similar levels of cell-cell communication. In contrast, cells giving rise to undifferentiated carcinomas, as well as cells harvested from undifferentiated carcinomas, exhibited very low levels or no homologous or heterologous cell-cell communication. Cell populations exhibiting distinctly different communication phenotypes were evaluated by Northern blot analysis for expression of connexins (Cx 26, 32 and 43) and E-cadherin. Neither communicating nor non-communicating cells expressed connexin 32. Those cell populations, which established functional gap junctions, expressed E-cadherin as well as connexin 26 and/or 43. In contrast, those cell populations that lacked the ability to communicate universally lacked expression of E-cadherin, and a quarter also lacked expression of detectable levels of connexin.     

焊接工艺参数对Q460GJC钢热影响区最高硬度的影响

焊接热影响区最高硬度试验能够用于评价钢板的抗冷裂纹性能。研究了焊接工艺对20 mm厚新型建筑用高性能结构钢Q460GJC的热影响区最高硬度的影响,结果表明:当选用较小线能量(9.0~10.0kJ/cm)的焊接工艺参数时,Q460GJC钢热影响区的最高硬度都大于350 HV,冷裂纹倾向较大;当选用较大线能量(14.5~15.5 kJ/cm)的焊接工艺参数时,Q460GJC钢热影响区的最高硬度都小于350HV,冷裂纹倾向降低。     

Transcellular retrograde labeling of radial glial cells with WGA-HRP and DiI in neonatal rat and hamster

Topographically distinct populations of radial glial cells in the diencephalon and mesencephalon of neonatal rats and hamsters were transcellularly labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and with the lipophilic tracer DiI. A comparison of the histological distribution of the two tracers is suggestive of two different mechanisms of transcellular labeling. Intraocular injections of WGA-HRP resulted in the uptake of exogenously applied WGA-HRP by retinal ganglion cells, followed by anterograde axonal transport and exocytosis within the optic target nuclei. In addition to the transneuronal labeling, which is typical of such injections, we observed the transcellular labeling of the processes and somata of radial glial cells that were topographically associated with the terminal fields of the labeled axons. Similar transcellular labeling of radial glial cells associated with the axon terminal fields of the colliculogeniculate projection to the medial geniculate nucleus was observed following injections of WGA-HRP in the inferior colliculus. The transcellular labeling within the radial glial cells was discontinuous and somatopetally concentrated, indicating the existence of a retrograde active transport mechanism within the radial glial processes subsequent to its uptake following release of tracer from axons. This type of labeling can be referred to as transcellular retrograde glioplasmic transport. In contrast, DiI was used as a tracer through its capacity to diffuse within the plasmalemma. Topographically distinct populations of radial glial cells were transcellularly labeled following placements of DiI in the retina, inferior colliculus, or dorsal thalamus of fixed brains. The radial processes of labeled radial glial cells consistently extended into regions that also contained labeled axons. It is likely that the transcellular radial glial labeling with DiI occurred via transmembranous diffusion. These data indicate that a close structural and functional relation exists between axons and glial cells in the developing brain.     

Gap junctional communication between murine macrophages and intestinal epithelial cell lines

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.     

Q345GJC钢板裂纹原因分析及控制

取样分析了高层建筑结构钢Q345GJC钢板的裂纹原因,结果表明,Q345GJC钢板上的裂纹是由于铸坯上产生的皮下裂纹所致,而且微合金化元素添加量及种类越多,连铸坯的裂纹敏感性越强。采取措施后,有效控制了Q345GJC钢板的裂纹发生率。     

Afferent and efferent connections of the parapineal organ in lampreys: a tract tracing and immunocytochemical study

The neural connections of the parapineal organ of two species of lampreys were studied with the fluorescent dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) and with immunocytochemistry. The lamprey parapineal organ consists of a vesicle and a ganglion that are connected to the left habenula. Labeling experiments included the application of DiI to the parapineal organ, left and right fasciculus retroflexus, left habenula, and the left pretectal region. Afferent parapineal fibers run in the left fasciculus retroflexus to the interpeduncular nucleus. The parapineal fibers of this fascicle arose from parapineal ganglion cells, whereas DiI application to the left habenula labeled both neurons of this ganglion and bipolar cells in the parapineal vesicle. Efferent neurons were observed in the left habenula, and bilaterally in the subhippocampal nucleus and the dorsal pretectum. Labeling with DiI also revealed a hippocampal projection. Immunocytochemical study of the parapineal vesicle revealed serotonin-immunoreactive cells in both species of lamprey, as well as substance P-immunoreactive (SP-ir) cells in sea lamprey and choline acetyltransferase-immunoreactive (ChAT-ir) cells in the river lamprey. The SP-ir cells and ChAT-ir cells formed a rich neuropil in the parapineal ganglion. Calretinin-ir cells were numerous in the ganglion. Neuropeptide Y-immunoreactive and gamma-aminobutyric acid-immunoreactive efferent fibers were observed in the parapineal organ. Neuropeptide Y-immunoreactive fibers originate in the subhippocampal nucleus, whereas gamma-aminobutyric acid-immunoreactive fibers might also arise in the pretectal nucleus. A few galanin-ir fibers were observed. These results indicate that the parapineal connections are completely different from those of the pineal organ. The possible homology between parapineal organs of vertebrates is discussed.     

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway

The projections of enteric neurons to the circular muscle of the guinea pig gastric corpus were investigated systematically by using the retrogradely transported fluorescent carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), applied to the muscle layer or myenteric plexus in vitro. DiI-labeled motor neuron cell bodies were located up to 6.3 mm aboral, 17 mm oral, and up to 20 mm circumferential to the DiI application site. Labeled nerve fibers ran for long distances from the DiI application site toward the greater and lesser curvatures, where they coursed parallel to the bundles of the "gastric sling" muscle. The majority of labeled cells were located toward the lesser curvature of the stomach. Nerve cell bodies that were aboral to the DiI application site were usually small, immunoreactive for choline acetyltransferase, and, thus, were likely to be excitatory motor neurons. Neurons that were located orally were larger, fewer in number, and immunoreactive for nitric oxide synthase and, thus, were likely to be inhibitory motor neurons. Application of DiI directly to the myenteric plexus filled neurons up to 15 mm aborally and up to 21 mm orally but labeled few neurons circumferentially. All nerve cells that were filled from either the circular muscle or the myenteric plexus had Dogiel type I morphological features. These results demonstrate a clear polarity of projection of inhibitory and excitatory motor neurons and a functionally continuous innervation of the circular and gastric sling muscle layers. Nonmotor neurons in the myenteric plexus were demonstrated, but neurons with Dogiel type II morphological features are apparently absent.     

In situ detection of PCR-amplified metalloproteinase cDNAs, their inhibitors and human papillomavirus transcripts in cervical carcinoma cell lines

Current fluorescence-based adhesion assays that use a 96-well plate format rely on the assumption that the fluorescent label does not significantly leak from the cells. Thus, we evaluated a calcein-based, in vitro adhesion assay in 96-well plates using five different types of leukocytes (HL60 cells, human neutrophils, rat neutrophils, mouse progenitor T cells and EL4 cells). Each cell type leaked calcein at a different rate, with the highest rates found for rat neutrophils and progenitor T cells, which lost as much as 20%-40% of the label within 90 min, the time required to complete the assay. Thus, we developed a procedure to measure the dye leakage rate during the assay in order to obtain a correction factor, which was then used to calculate the "true" number of adherent cells. Data for the adhesion of FTF1 cells to endothelial monolayers, after correction for calcein leakage, deviated less than 10% of adhesion data obtained with a well-established 51Cr-based assay.     

Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia

A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (DiI) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytohemagglutinine (PHA) or lipoprotein-deficient serum (LPDS). LPDS-treated monocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for DiI LDL binding by about 40%. In competition with unlabeled LDL, DiI LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of DiI LDL were also similar to 125I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for DiI LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or DiI LDL at 4 degrees C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal DiI LDL for 4 degrees C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4 degrees C DiI LDL binding, but at 20 degrees C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isoforms and lipoprotein[a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of DiI LDL or familial defective apolipoprotein B-100 was detected.(ABSTRACT TRUNCATED AT 400 WORDS)     

Imaging the mitochondrial permeability transition pore in intact cells

The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.     

Confocal microscopy of human lens membranes in aged normal and nuclear cataracts

PURPOSE: To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses. METHODS: Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus. RESULTS: DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses. CONCLUSIONS: The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.     

高层建筑结构用Q345GJC钢板的研制

介绍了高层建筑结构用Q345GJC钢设计的化学成分、加热工艺及控轧工艺,试制结果表明:Q345GJC钢设计的化学成分及生产工艺合理,其各项性能指标满足国标要求,试制获得成功。     

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.     

建筑结构用钢Q345GJC的研制与开发

利用铌微合金化技术及控制轧制、控制冷却工艺,邯钢自主研发了高层建筑结构用钢Q345GJC。Q345GJC钢质纯净,硫、磷含量低,钢板焊接性能优良,具有高强度、良好的韧性、良好的表面和内部质量,各项指标全部符合标准要求。     

Adhesion and cytosolic dye transfer between macrophages and intestinal epithelial cells

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.     

Event-related EEG desynchronization and synchronization during an auditory memory task

Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins in Xenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.     

Protection of herpes simplex virus thymidine kinase-transduced cells from ganciclovir-mediated cytotoxicity by bystander cells: the Good Samaritan effect

Although considerable attention has been directed in the field of gene therapy toward elucidating the mechanism by which a transduced cell could kill a bystander cell, little is known about how bystander cells may affect transduced cells. We hypothesized that bystander cells, particularly if they were capable of gap junctional communication, could protect cells transduced with the herpes simplex virus thymidine kinase (HSV-TK) from ganciclovir (GCV)-induced cytotoxicity. To test this hypothesis, we used a rat hepatocyte cell line (WB) that can carry out efficient gap junctional communication, a WB clone transduced with HSV-TK (WB-TK), and a communication-incompetent subclone of WB cells (aB1). We cocultured WB-TK cells with either WB or aB1 cells, treated them with GCV, and then plated the cells into selective media that permitted us to quantify independently the surviving fraction of WB-TK cells or bystander cells. We found that WB bystander cells conferred up to a 1000-fold protection on WB-TK cells treated with GCV. aB1 cells conferred detectable, but significantly less, protection. These findings demonstrate that herpes simplex virus thymidine kinase-transduced cells can be significantly protected by bystander cells, particularly those that can carry out gap junctional communication. Whether this "Good Samaritan" effect improves the overall efficacy of gene therapy, by prolonging the survival of the source of toxic metabolites, or decreases effectiveness by increasing the survival of transduced cells will need to be determined in vivo.