Compositions and Positional Distributions of Fatty Acids in Phospholipids from Leaves of Chilling-Sensitive and Chilling-Resistant Plants

The compositions and positional distributions of fatty acidsin the major leaf phospholipids of phosphatidylglycerol, phosphatidylcholineand phosphatidylethanolamine were analyzed by gas-liquid chromatographyand enzymic hydrolysis, and chilling-sensitive and chilling-resistantplants were comparcd with respect to the relative contents ofpalmitic and trans-³-hexadecenoic acids in the separated phospholipids.A distinct difference between these plants was found in thefatty acid compositions of phosphatidylglycerol, in which thesum of palmitic and trans-³-hexadecenoic acids ranged from 60to 78% of the total fatty acids in 8 species of chilling-sensitiveplants, and from 50 to 57% in 11 species of chilling-resistantplants. The only exception among the chilling sensitive plantsin this respect was the tomato, in which the sum of palmiticand trans-³-hexadecenic acids in phosphatidylglycerol amountedto 54%. The fatty acid compositions and the positional distributionsof fatty acids in phosphatidylglycerol suggest that the occurrenceof high proportions of dipalmitoyl and 1-palmitoyl-2-(trans-³-hexadecenoyl)species in this lipid is correlated with the susceptibilityto chilling of the leaves of higher plants. In the compositionsand positional distributions of fatty acids in phosphatidylcholineand phosphatidylethanolamine, no difference was found betweenthe chilling-sensitive and chilling-resistant plants. ¹ Present address: Department of Biology, Faculty of Science,Universityof Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received May 21, 1982; Accepted June 25, 1982)     

Protein phosphatase 2A in stretch-induced endothelial cell proliferation

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459–466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.     

Promoting effects of 105K glycoprotein on cell proliferation in chicken lymphoblastoid cell lines

Abstract The promoting effects on cell proliferation of the 105K glycoprotein (105Kgp), purified from sera of chickens to which a Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C (MSB1-41C), had been transplanted, were examined using culture cells from various sources. The MSB1-41C line as well as the other chicken lymphoblastoid cell lines examined were sensitive to the 105Kgp. The growth-promoting effects of 105Kgp showed a biphasic pattern depending upon the amount of 105Kgp added into the culture medium.
These findings indicate that the 105Kgp may be a promoting factor for chicken growing cells, especially lymphoblastoid cell lines.     

Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells.

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [(3)H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca(2+)](i) increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca(2+) responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca(2+) system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.     

Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp. A1

The NAD kinase gene (nadK) of Sphingomonas sp. A1 was cloned and then overexpressed in Escherichia coli, and the gene product (NadK) was purified from the E. coli cells through five steps with a 25% yield of activity. NadK was a homodimer of 32 kDa subunits, utilized ATP or other nucleoside triphosphates, but not inorganic polyphosphates, as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 8.0 and 50-55 degrees C, and was designated as ATP-NAD kinase (NadK). NadK showed no NADH kinase activity and was slightly inhibited by NADP(H). Precursors for NAD biosynthesis such as quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinic acid had no effect on the NadK activity, as observed in the cases of the NAD kinases of Micrococcus flavus, Mycobacterium tuberculosis, and E. coli. Taken together with the report that the NAD kinase of Bacillus subtilis is activated by quinolinic acid [J. Bacteriol. 185 (2003) 4844], it is indicated that the regulatory patterns of NAD kinases differ even among bacterial NAD kinases.     

Cdc2 kinase-dependent disassembly of endoplasmic reticulum (ER) exit sites inhibits ER-to-Golgi vesicular transport during mitosis

We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP–labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis.     

Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.     

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.     

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.