Indomethacin inhibits thrombin-, but not thyroxin-stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum. Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM-10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10 uM indomethacin.     

Cytokeratin expression in simple epithelia

To study the regulation of the expression of cytokeratins characteristic of simple epithelia, i.e., human cytokeratins nos. 7, 8, 18, and 19, we prepared several cDNA clones coding for these proteins and their bovine counterparts. In the present study, we describe a cDNA clone of the mRNA coding for human cytokeratin no. 18, which was isolated from an expression library using the monoclonal antibody, KG 8.13. This clone (756 nucleotides, excluding the polyA portion), encodes approximately one-half of the mRNA (approximately 1.4 kb), identifies one mRNA band in Northern-hybridization blots, and specifically selects one mRNA species coding for cytokeratin no. 18, as demonstrated by translation in vitro. Comparison of the deduced amino acid sequence--confirmed by direct amino-acid-sequence analyses of some polypeptide fragments produced by cleavage with cyanogen bromide--indicated that cytokeratin no. 18 is a member of the acidic (type I) subfamily of cytokeratins. It has only limited sequence homologies in common with other intermediate-sized filament proteins, and these are essentially restricted to certain domains of the alpha-helical rod portion. The carboxyterminal tail sequence does not contain glycine-rich elements, thus distinguishing this cytokeratin from those acidic (type I) cytokeratins that are characterized by this feature. The similarities and differences between cytokeratin no. 18 and previously described epidermal cytokeratins are discussed in relation to the differences in the stability of the complexes which this cytokeratin forms with basic (type II) cytokeratins, as well as in relation to possible functional differences of cytokeratins in simple and stratified epithelia.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

The amount and nature of glutathione transferases in rat liver microsomes determined by immunochemical methods

The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 ± 0.6 and 1.5 ± 0.1 μg/mg microsomal protein, respectively. These levels are 10-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 31 ± 6 μg/mg microsomal protein.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Dynamics of the cytoskeleton in Amoeba proteus

Fluorescein-labeled muscle actin was microinjected into Amoeba proteus and followed during intracellular redistribution by means of the image-intensification technique. The fully polymerization-competent protein becomes part of the endogenous actomyosin system undergoing dynamic changes over time periods of several hours. Single-frame analysis of long-term sequences enabled the direct demonstration of both the contractile activities and morphological transformations of microfilaments in normally locomoting, immobilized and phagocytozing specimens. In normally locomoting cells the filament layer undergoes continuous changes in spatial distribution depending on the actual pattern of cytoplasmic streaming and cell shape. The highest degree of differentiation is always maintained in the intermediate region between the front and the uroid, thus indicating this segment of the cortex to be the most important site in generating motive force for pseudopodium formation and ameboid movement. In immobilized cells contracted by the application of ruthenium red or relaxed by different anesthetics, the filament layer forms a continuous thick sheath beneath the cell surface or becomes completely disintegrated. In phagocytozing cells the local polymerization of actin at the tip of pseudopodia forming the food-cup and around the nascent phagosome points to a significant participation of the actomyosin system in the process of capturing and constricting prey organisms. Although our results provide clear evidence for the overall importance of motive force generation according to the hydraulic pressure theory, some motile phenomena exist in Amoeba proteus that cannot exclusively be explained by this mechanism.