Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

Heme prosthetic group required for acetylation of prostaglandin H synthase by aspirin

The ability of aspirin to acetylate PGH synthase was determined by reacting [3H-acetyl]-aspirin with purified enzyme followed by high pressure liquid chromatography analysis of the protein components of the reaction mixture. Heme-reconstituted enzyme incorporated approximately one acetyl group per 70-kDa subunit, whereas apoprotein incorporated 0.1 acetyl group per subunit. The ability of the heme prosthetic group to enhance acetylation of the protein was correlated with its ability to protect the Arg253-Gly254 peptide bond from cleavage by trypsin. Thus, heme-induced alteration of protein conformation may contribute to the enhanced labeling of Ser506 by aspirin. The present results indicate that irreversible inactivation of prostaglandin H synthase by aspirin occurs only when the heme prosthetic group is bound to the protein. Considering its short in vivo half-life, it is likely that aspirin inactivates only the steady-state fraction of PGH synthase in a cell that is active but not newly synthesized apoprotein. This may contribute to the differential kinetics of inactivation and recovery of PGH synthase activity in platelets and vascular endothelial cells after administration of low dose aspirin as a prophylactic agent against cardiovascular disease.     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

Tolerance of tannin by the shiitake mushroom,Lentinus edodes

Summary Tannin at 1% (w/v) did not inhibit the growth ofLentinus edodes, but did inhibitPleuroius florida, P. sajor-caju, P. cystidosus, Agaricus bisporus andVolvariella volvacea. The inhibition was not due to its acidity.

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Tolérance de Lentinus edodes aux ajouts de tannin

Résumé Le tannin à la concentration de 1% (p/v) n'inhibe pas la croissance deLentinus edodes, mais inhibe celle dePleurotus florida, P. sajor-caju, P. cystidosus, Agaricus bisporus, etVolvariella volvacea. L'inhibition n'est pas due à son acidité.

     

Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.     

The pig as an intermediate host for Taiwan Taenia infection

Eggs (1000-100,000/animal) of Taiwan Taenia were inoculated per os into 14 Small-Ear-Miniature (SEM), 19 Landrace-Small-Ear-Miniature (L-SEM), and 5 Duroc-Yorkshire-Landrace (DYL) pigs. These animals were sacrificed 7-107 days after infection. Thirty-four pigs were found to be infected with Taiwan Taenia cysticerci and the infection rates of SEM, L-SEM, and DYL were 86%, 89% and 100% respectively. The cysticerci recovery rates of SEM, L-SEM and DYL pigs were 27.2%, 1.7% and 0.27% respectively. Cysticerci were recovered only from the livers and none were found in muscles, viscera or other parts of the carcasses. More cysticerci were located in the liver parenchyma (71%) than on the liver surface (29%). Taiwan Taenia cysticerci were smaller than those of classical T. saginata or T. solium. Moreover, Taiwan Taenia cysticerci had 2 rows of rudimentary hooklets on the scolex. The results of this study indicate that young pigs are good intermediate hosts for Taiwan Taenia and that the SEM pig is a satisfactory host for experimental studies with this tapeworm. These results were similar to other studies with different geographic strains of the T. saginata-like tapeworm in the Far East. These strains appear to be the same and possibly a new species.     

Mechanism of gating of T-type calcium channels

We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner.     

ELISA measurement of LDL receptors

An enzyme-linked immunosorbent assay was developed for measurement of low density lipoprotein (LDL) receptors. A monospecific polyclonal antibody to LDL receptor purified from rat liver that reacted with rat, mouse, canine, and human LDL receptor was used. With this assay, LDL receptors could be measured on 2-4 x 10(5) adherent cells and 1.0 x 10(5) cells in suspension, although results were more variable with cell suspensions. Membranes from a variety of receptor-rich and receptor-poor tissues could be assayed directly after adherence of the membranes to the ELISA plate by an overnight incubation. In some instances, the quality of the assay was improved by first solubilizing the membranes. The sensitivity of the assay is such that between 0.15 and 2 micrograms of membrane protein is required. This could be obtained from leukocytes in a modest (20-30 ml) quantity of human blood. The assay was used to demonstrate the rapid down-regulation of LDL receptors in human mononuclear leukocytes in response to a cholesterol-containing meal. Overall, the results support the use of ELISA technology to measure LDL receptors, particularly for physiologic studies.