Occurrence of pre-MBT synthesis of caspase-8 mRNA and activation of caspase-8 prior to execution of SAMDC (S-adenosylmethionine decarboxylase)-induced, but not p53-induced, apoptosis in Xenopus late blastulae

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.     

Regulation of insulin receptor internalization in vascular endothelial cells by insulin and phorbol ester

Phorbol 12-myristate 13-acetate (PMA) was used to examine the role of insulin receptor phosphorylation in the regulation of insulin receptor internalization in vascular endothelial cells. Association of 125I-insulin in rat capillary and bovine aortic endothelial cells preincubated with PMA was increased by 80 and 64% over control, respectively. The increase was due to enhanced 125I-insulin internalization as opposed to an effect on surface-bound hormone. PMA had no significant effect on 125I-insulin degradation or on release of internalized insulin from the cells. Internalization of 125I-labeled insulin receptor was determined by the resistance of labeled receptor to trypsinization. At 10 degrees C, nearly all of the labeled receptor was sensitive to removal by trypsin, indicating that it was exposed on the cell surface. Exposure of labeled cells to insulin (100 nM) at 37 degrees C resulted in the rapid appearance of trypsin-resistant insulin receptor, indicating receptor internalization. Steady state for receptor internalization was attained at 10-15 min. When surfaced-labeled cells were preincubated with PMA at 37 degrees C, the rate of insulin receptor internalization was increased by 3.6 +/- 0.2-fold and 2.1 +/- 0.5-fold at 1 and 5 min of insulin exposure, respectively (ED50 at 16 nM PMA). This effect of PMA was associated with an increase in serine phosphorylation of the insulin receptor. Thus, PMA increased insulin internalization in the endothelial cells by modulating the insulin-induced internalization of the receptor. The additive effects of PMA and insulin on insulin receptor phosphorylation suggest that the phorbol ester and insulin act via independent signaling mechanisms.     

Selective inhibition of membrane fusion events in echinoderm gametes and embryos by halenaquinol sulfate.

Halenaquinol sulfate, a hydroquinone sulfate obtained from the sponge Xestospongia sapra, prevented cell membrane fusion events of echinaderm gametes but did not affect early embryonic development of fertilized eggs up to the gastrula stage. However, halenaquinol sulfate inhibited secretion of hatching enzyme, resulting in the formation of gastrulae that were surrounded by the fertilization envelope. Therefore, the use of halenaquinol sulfate offers a unique opportunity to analyze the role of secretory events in complex populations of cells without affecting other cellular functions.     

Glucosamine-derived phospholipids in Escherichia coli. Structure and chemical modification of a triacyl glucosamine 1-phosphate found in a phosphatidylglycerol-deficient mutant

Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate novel glucosamine-derived phospholipids. We previously demonstrated that the simplest of these substance (lipid X) is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3 (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379-7385). We now report the structural characterization of a triacylglucosamine 1-phosphate (designated lipid Y) that is also found in these mutants. Hydrolyzates of Y contain 2 mol of beta-hydroxymyristate and 1 mol of palmitate/mol of glucosamine. In the lipid, one of the beta-hydroxymyristates is amide-linked at position 2, while the two other fatty acyl groups are ester-linked. Fast atom bombardment mass spectrometry is used to confirm that Y is a monosaccharide derivative and that the molecular weight of Y as the free acid (C50H96NO13P) is 950.29. Analysis of Y by proton NMR spectroscopy at 200 MHz reveals that the anomeric configuration is alpha. Further, one of the esterified fatty acid residues is attached to the 3 OH of the sugar, while the second is linked to an OH moiety of a hydroxymyristate. The 4 and 6 OH groups of the sugar are unsubstituted, as in E. coli lipid X. To establish the precise location of each esterified fatty acyl residue, we subjected Y to a very mild alkaline hydrolysis in the presence of triethylamine. This resulted in the selective removal of a single hydroxymyristoyl group. The triethylamine-treated derivative (lipid Y) has a molecular weight of 723. NMR spectroscopy of Y shows that the 3 OH of the sugar is no longer substituted, while the beta OH of the remaining amide-linked hydroxymyristate is still esterified with palmitate. On the basis of these findings, we propose that lipid Y has the same fundamental structure as lipid X, except for the additional presence of a palmitoyl moiety on the N-linked hydroxymyristate. Presumably, lipid Y is synthesized from X by a selective acylation reaction.     

Changes in prostaglandin levels in cultures of SV40 -transformed macrophage cell lines in relation to their phenotypic expression

A radioimmunoassay was performed to determine the total amounts of the A, B, and E series prostaglandins (prostaglandins) in culture fluids of simian virus 40 (SV40)-transformed mouse clonal macrophages, line 28-12, and its subline, 28-12 (Ara). When the proportion of phagocytic cells in confluent 28-12 cell cultures increased, the prostaglandin levels in the culture fluids decreased. On the other hand, stably phagocytic 28-12 (Ara) cells, which were derived from 28-12 cells and which had a reduced growth rate, did not release prostaglandins under the usual culture conditions; however, when they were treated with lipopolysaccharide or streptococcal preparation OK-432, large amounts of prostaglandins were released. In contrast, nonphagocytic cell populations in the cultures of 28-12 cells were not responsive to the drug treatment. These results suggest that there is a correlation between the phenotypic change from the nonphagocytic to the phagocytic state and a decrease in prostaglandin levels in culture fluids, and indicate that phagocytic cells are responsive to prostaglandin inducers, whereas nonphagocytic cells are not.     

Inhibition of Intracellular Multiplication of Adenovirus by Interaction between Infected and Uninfected Cells

The possibility that virus multiplication may be inhibited by interaction of infected cells with uninfected cells was tested by experiments, using human adenovirus type 12 (Ad 12). Permissive human cells (human embryonic kidney = HEK, KB or HeLa) were infected and seeded on uninfected or infected “nonpermissive” cell (human embryonic lung = HEL) monolayers, and virus yields or proportions of viral antigen-synthesizing cells were compared with each other. Both the virus yields and the proportions of viral antigen-positive cells were not reduced significantly when seeded on infected HEL cells, while when seeded on uninfected HEL cells both of them were reduced remarkably, compared with the yield and the proportion of controls seeded on glass. Similar results were obtained regardless of the type of permissive cells, HEK, KB, or HeLa. Similar reduction of the yield was observed when seeded on HEL cells infected with Ad 12 inactivated by heat or by antiserum, and partial reduction was observed when seeded on HEL cells infected with UV-inactivated Ad 12, depending on the extent of UV dosis. These experiments showed that intracellular virus multiplication may be inhibited by interaction of infected cells with uninfected cells, and this may be due to the difference in the cell surface structure.     

The cognition-enhancer nefiracetam is protective in BDNF-independent neuronal cell death under the serum-free condition.

Cortical cells from embryonic mice (E17) showed a rapid cell-death under the serum-free condition. The addition of nefiracetam at 0.1-10 microM increased the survival activity, while aniracetam at the same concentrations did not. The cell death was characterized to be apoptotic, since dead cells showed nuclear condensation, fragmentation, and TUNEL-positive staining. The nefiracetam-induced anti-apoptotic activity was completely blocked by 1 microM nifedipine or omega-conotoxin GVIA, and partially by 1 microM verapamil. These results suggest that the reported anti-amnesic action of nefiracetam in ischemic animals may be partly attributed to the neuroprotective action.     

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells.

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.     

Clonal Variation in the Sprouting Pattern of the Tubers in Eleocharis kuroguwai, a Cyperaceous Weed, with Special Reference to its Perennation Strategy

Abstract Clonal variation in the sprouting pattern of tubers in Eleocharis kuroguwai Ohwi was studied, using 39 clones collected within a longitudinally narrow belt from Wakasa Bay to the Kii Peninsula of Japan and one of "Ohkuroguwai", a cultivated form.
The sprouting pattern of tubers and the relationship between sprouting time and other tuber characteristics varied noticeably among clones. Based on the polygonal graphs of × (mean), (standard deviation), g₁ (skewness) and á (kurtosis) of the sprouting time, the sprouting pattern of 40 clones could be divided into the following three groups. Group A: and α were small, but g₁ and a' were generally great; almost all tubers sprouted early and simultaneously. Group B: and α were greater, but g₁ and a' were smaller than Group A; the majority of tubers sprouted quickly, but the rest of the tubers sprouted gradually. Group C: and α were very large, g₁ was small, but a' showed a comparatively large negative value; all tubers sprouted gradually over a long period.
Groups A and B were represented by the irrigation pond group from the Banshu Plain and by the Ohshima Island group in the paddy fields of the southern extremity of the Kii Peninsula. Clones from the paddy field group and from the plain group, both referable to typical rstrategists, exhibited various sprouting patterns of tubers.     

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

Key message

The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.

Abstract

Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.