Further studies on the relationship between tyrosine supply and catecholamine production in cultured adrenal chromaffin cells.

To elucidate a possible role of tyrosine supply as a factor modulating catecholamine biosynthesis in the adrenergic cell, the transport of [14C]tyrosine into cultured bovine adrenal chromaffin cells was first examined, and the relationship between [14C]tyrosine transport and [14C]catecholamine formation was then investigated. Under the conditions which were routinely employed to determine the rate of catecholamine biosynthesis, tyrosine was taken up into the cells in a manner independent of extracellular Na+ and Ca2+, and this uptake was also insensitive to ouabain and various metabolic inhibitors. The stimulation of these cells with high K+ and other secretagogues caused no significant alteration in the uptake. While, tyrosine transport was markedly inhibited by tyrosine analogues and other L-aromatic amino acids, and this inhibition was accompanied by the reduction of [14C]catecholamine formation. In contrast, tyrosine transport was markedly enhanced by flavone, and this enhancement was also accompanied by the augmentation of catecholamine production under the same experimental conditions. These results seem to indicate that the transport of tyrosine into the cells may be closely related to catecholamine formation within the cells, thus providing an evidence for a possible role of tyrosine supply as one of the factors affecting catecholamine production in the adrenal chromaffin cell.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Promotive Effect of C18-Unsaturated Fatty Acids on the Abscission of Bean Petiole Explants

The abscission-promoting effects of C₁₈-unsaturated fatty acidswere studied in bean (Phaseolus vulgaris L. cv. Masterpiece)petiole explants with the junction between the petiole and thepulvinus in the primary leaves in the light. Linolenic, linoleicand oleic acids promoted the abscission of the explants in thelight. Linolenic acid was the most effective among the compoundstested and its promotive effect was evident without any accompanyingincrease in the production of ethylene from the explants, ascompared with non-treated explants. Linolenic acid is easilyconverted to its hydroperoxide during the incubation with explants,as indicated by the formation of the conjugated diene and thegeneration of ethane. The production of ethylene from the explantstreated with linolenic acid was completely inhibited by theaddition of aminoethoxyvi-nylglycine (AVG), but large amountsof ethane were still generated. The promotive effect of linolenicacid was almost eliminated by the addition of scavengers offree radicals. Hydrogen peroxide and tert-butyl hydroperoxidepromoted abscission in the light. From these results, we concludedthat the abscission-promoting effect of linolenic acid are notmediated by the effect of ethylene but by the effect of itshydroperoxide, while the well-established pathway for the biosynthesisof ethylene from S-adenosylmethionine to ethylene, via 1-aminocyclopropane-l-carboxylicacid (ACC), was apparently operative. (Received May 1, 1991; Accepted July 10, 1991)     

Overexpression of a homeodomain protein confers axis-forming activity to uncommitted Xenopus embryonic cells.

The anteroposterior character of mesoderm induced by a peptide growth factor (XTC-MIF) was tested by transplantation into host Xenopus gastrulae. Both retinoic acid and a homeodomain protein were able to override the anteriorizing effect of the growth factor. Microinjection of a posteriorly expressed homeobox mRNA can respecify anteroposterior identity, transforming head mesoderm into tail-inducing mesoderm. Unexpectedly, overexpression of XIHbox 6 protein in the transplanted cells, without addition of growth factors, caused the formation of tail-like structures. The cells overexpressing XIHbox 6 were able to recruit cells from the host into the secondary axis. The results suggest that vertebrate homeodomain proteins are part of the biochemical pathway leading to the generation of the body axis.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

Comparative study of embryonic thermoresistance of two inbred strains of the medaka (Oryzias latipes)

Summary By using inbred strains (HO4C and HB32C) of the medaka,Oryzias latipes, the involvement of genetic factor(s) in the determination of thermoresistance of fish was investigated. The thermoresistance of embryos of the medaka was quantitated by the fraction of the embryos surviving 1 day after heat treatment. At early stages of development (st. 13 and st. 20–21), the HO4C strain was more resistant than the HB32C strain. At st. 20–21, the HO4C strain was more resistant than the HB32C strain at all temperatures used (42, 43, and 44°C). At later stages of development (st. 27 and st. 32), however, the HB32C strain was more resistant than the HO4C strain.The results of genetic cross experiments raised the following possibilities; the thermoresistance of embryos at early developmental stages can be lowered by some factor(s) inherited in the HO4C strain and/or increased by those in the HB32C strain. By contrast, the sensitivity of embryos at later stages of development was not affected by factor(s) of their parents, but by their own genetic constitution.     

Phosphorylation of Delta Sleep-Inducing Peptide (DSIP) by casein kinase II in vitro

A phosphorylated analogue of DSIP at Ser⁷ has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent K_m and V_max values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.     

Monoclonal antibodies that depress a specific subset of multiple components of the glutathione-induced response ofHydra

Summary Reduced glutathione evokes a feeding response, the tentacle-ball formation inHydra japonica. This response consists of at least 5 components (R1–R5). We raised 6 monoclonal antibodies (mAbs), each of which depressed a specific subset of these components, and we also examined the immunocytochemical localization of antigens with these mAbs at light microscopic level. The 2 mAbs that depressed R2 and R4 bound to the cnidocils of the desmoneme and the stenotele nematocytes; the 3 mAbs that depressed R5 bound to the apical surface adjacent to the cnidocils of the nematocytes; and the 2 mAbs that depressed R1 and R3 bound to the apical spot structures of unidentified cells in the ectoderm.Together with the specificity of the action of the mAbs on the behavioral response, the correspondence between the effects on the response and the structures visualized with these mAbs suggests that these structures include components of the receptor-effector system relevant to chemoreception.     

Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.