Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Stimulation of the Flowering of Pharbitis nil Chois. by Gibberellin A3 : Time Dependent Action at the Apex

Gibberellin A₃ (GA₃) stimulated flowering when it was appliedto the shoot apex of seedlings of Pharbitis nil, dwarf strainKidachi; but, not when it was applied to the cotyledons. GA₃applied to the plumule before or shortly after the start ofan inductive dark period promoted both flowering and shoot elongation;but, the later the time of application during the dark periodless the promotion of flowering, although marked promotion ofshoot elongation always took place. The variation with time in the response of flowering to GA₃indicates that early floral processes at the apex are stimulatedby GA₃, but that subsequent processes are insensitive to it.The early processes of floral stimulus produced by a 16 hr inductivedark period probably are completed within 20 hr at 28°Cafter the end of the dark period. At low temperatures, suchas 15 and 20°C, early floral processes continued for morethan 40 hr. When cotyledons were removed at various times, the export ofthe floral stimulus to the shoot apex was apparent within hoursof the generation of the floral stimulus in the cotyledons,which started with the passage of the critical 9-hr dark period. (Received February 18, 1981; Accepted March 24, 1981)     

Hydrocephalus in suckling rats infected intracerebrally with mouse hepatitis virus, MHV-A59

After intracerebral inoculation of mouse hepatitis virus, MHV-A59 strain, into 3- to 5-day-old Wistar rats, some survivors at 14 days postinoculation (p.i.) were found to lack the cerebral cortex and to have an accumulation of considerable amount of cerebrospinal fluid. The virus titer in the brain increased exponentially after inoculation, reaching a maximum 4 to 6 days p.i. when immunofluorescence revealed virus-specific antigen within neurons in the cerebral cortex. A small amount of infectious virus was also detectable 14 days p.i. when the cerebral anomaly was evident. This brain malformation causing hydrocephalus was due to cerebral damage by viral infection.     

Synthesis of 2,2,2-trichloroethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside,and its reaction with glycosyl halides.

2,2,2-trichloroethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (9) was synthesized in 6 steps from the readily available 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranose in 25% overall yield by employing the stannyl method for the regioselective activation of hydroxyl groups. Dibenzyl ether 9 was then glycosylated with appropriate glycosyl donors to afford lactosamine and chitobiose derivatives in good yield.     

Nuclear magnetic resonance studies of hemoproteins. VIII. Proton NMR studies of the binding of various nitrogenous ligands to ferric cytochrome c.

The structure of the heme environment of horse heart ferric cytochrome c was examined in the presence of various nitrogenous bases at several temperatures with the aid of hyperfine shifted proton NMR spectra at 220 MHz. The resonance positions and line widths of the signals for the peripheral methyl groups of the heme exhibited distinctive features of its low-spin state characteristic of each external ligand. In the imidazole complex of ferric cytochrome c, remarkable line sharpening of the heme-linked proton signals was encountered on raising the temperature. This may be related to the apoprotein perturbation on the binding of external ligand to the heme iron. These spectral peculiarities were discussed in relation to the electronic structure of the heme, the basicity of the external ligand and the van der Waals contact interaction between heme side chains and apoprotein.     

Two glycosulfatases from the liver of a marine gastropod, Charonia lampas. Partial purification and properties.

Two glycosulfatases [EC 3.1.6.3], I and II, were purified 31.3- and 33.9-fold respectively, from a crude extract of the liver of Charonia lampas. The purification was carried out by the following chromatographic procedures; phosphocellulose, Sephadex G-150, Concanavalin A-Sepharose and isoelectric focussing. The enzyme preparations obtained were practically free from arylsulfatase [EC 3.1.6.1] contamination. Both glycosulfatases are probably glycoproteins differing in their carbohydrate moieties. The molecular weights of glycosulfatase I and II were estimated to be about 112,000 and 79,000 respectively. They had the same optimum pH of 5.5, and the same Km value of 25.0 mM for glucose 6-sulfate.     

Image-based parameter inference for epithelial mechanics

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

Critical requirement for the membrane-proximal cytosolic tyrosine residue for CD28-mediated costimulation in vivo

The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.