Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100–300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives.
Imaging with Zernike phase plates is increasingly being used in cryo-TEM tomography and cryo-EM single-particle applications. However, rapid ageing of the phase plates, together with the cost and effort in producing them, present serious obstacles to widespread adoption. We are experimenting with phase plates based on silicon chips that have thin windows; such phase plates could be mass-produced and made available at moderate cost. The windows are coated with conductive layers to reduce charging, and this considerably extends the useful life of the phase plates compared to traditional pure-carbon phase plates. However, a compromise must be reached between robustness and transmission through the phase-plate film. Details are given on testing phase-plate performance by means of imaging an amorphous thin film and evaluating the power spectra of the images.
D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.
Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor-oxaloacetate (OA) or alpha-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between 3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.
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