Multiple,concentration-dependent effects of sucrose,auxins and cytokinins in explant cultures of kale and tobacco

We describe complex multiple concentration dependencies for the response of isolated pith tissues to plant biologically active substances. Kale and tobacco stem pith explants were cultured on agar media containing combinations of sucrose, cytokinin [kinetin or benzyladenine (BA)] and auxin [indole-3-acetic acid (IAA) or naphthaleneacetic acid (NAA)]. Absorption of these components by explants and their effects on explant mass, contents of soluble proteins, starch and sugars, and activity of ADP-glucose pyrophosphorylase (AGPase) were studied in relation to their concentration. Up to ten pronounced statistically significant maxima (peaks or waves) were repeatedly detected in the dose–response curves over a concentration range of several logarithmic orders. Slight maxima were observed in the corresponding absorption curves. Pronounced maxima of sucrose absorption were induced by IAA and BA, and those of NAA absorption were induced by sucrose. Both types of multiple maxima (in dose–response and absorption curves) may be due to changes in concentration of intracellular solutes (sugars, auxins and cytokinins), thereby affecting metabolic processes that act as sinks for external solutes and elicit feedback appearance of maxima in absorption curves. Good correspondence between external concentrations at which maxima of different compared curves occur in addition to statistical significance of individual maxima and repeatability of experimental results supports the conclusion that the multiple maxima exhibited are genuine. We consider it possibile that the multiple maxima are associated with endopolyploidy or mixoploidy and/or epigenomic diversity of pith cells that show different sensitivities to biologically active solutes.     

7-Ketocholesterol Incorporation into Sphingolipid/Cholesterol-enriched (Lipid Raft) Domains Is Impaired by Vitamin E: A SPECIFIC ROLE FOR ��-TOCOPHEROL WITH CONSEQUENCES ON CELL DEATH*

Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike γ-tocopherol, the α-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, α-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with α-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of α-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by α-tocopherol, but not γ-tocopherol pretreatment.It has been suggested that cholesterol oxide-induced apoptosis is a key event in the initiation and progression of atherosclerosis lesions (1, 2). In the initial step of the disease, cholesterol oxides in modified low density lipoproteins were found to promote the death of endothelial cells lining the intravascular lumen (1, 2). In more advanced stages and as the atherosclerotic lesion progresses, cholesterol oxides could also contribute to the destruction of foam cells and vascular smooth muscle cells, to the formation of the lipid core, to the reduction of cell proliferation, and eventually to plaque destabilization (1, 2). Among cholesterol oxides that are mainly synthesized during oxidation of low density lipoproteins, 7-ketocholesterol is one of the most abundant in plasma and atherosclerotic lesions (3). Moreover, in a number of cell models, it has been established that 7-ketocholesterol is one of the cholesterol oxide derivatives with the highest pro-apoptotic potential (4, 5). The 7-ketocholesterol derivative associates preferentially with membrane lipid raft domains (6), which are characterized by the lateral packing of glycosphingolipids, sphingolipids, and cholesterol. Because of their insolubility in cold non-ionic detergents, rafts are also called detergent-resistant membranes (7). These structures are thought to be involved in cellular signaling mechanisms (8, 9). It is worthy of note that 7-ketocholesterol has been shown to induce cell death through inactivation of the phosphatidylinositol 3-kinase/Akt signaling pathway (10), which is known to be highly specific to lipid raft domains (9).Vitamin E is composed of closely related molecules, i.e. tocopherols and tocotrienols, which are each composed of four α, β, γ, and δ analogues. Although vitamin E was widely studied for its ability to prevent cellular damage by reactive oxygen species, it has recently been the subject of intense research for its putative, non-antioxidant functions (11, 12). Among the various forms of vitamin E, α-tocopherol is most abundant in the body as it is specifically recognized by the liver α-tocopherol transfer protein. Although several studies have shown that vitamin E has the ability to counteract the pro-apoptotic effect of 7-ketocholesterol in cultured cells (10, 13), the underlying molecular mechanism is unclear.In the present study the molecular mechanism involved in the vitamin E-mediated protection against apoptosis induced by 7-ketocholesterol was investigated on the well known A7R5 aortic smooth muscle cell model. It is reported here that α-tocopherol, but not γ-tocopherol, effectively protects the cells against 7-ketocholesterol-induced apoptosis when applied as a pretreatment before the addition of 7-ketocholesterol. Unlike γ-tocopherol, α-tocopherol was able to activate the Akt-PKB anti-apoptotic signaling pathway in the lipid raft domains (14), leading to phosphorylation and, thus, inactivation of Bad (15). Most importantly, the protective effect of α-tocopherol is shown to operate through its prior incorporation into the lipid raft domains of the plasma membrane, which leads to the subsequent exclusion and, thus, inactivation of 7-ketocholesterol.     

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.     

Demonstration of dimethylnonanoyl-CoA thioesterase activity in rat liver peroxisomes followed by purification and molecular cloning of the thioesterase involved.

Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic acid undergoes three cycles of beta-oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA (DMN-CoA) which is then transported to the mitochondria for full oxidation to CO(2) and H(2)O. In principle, this can be done via two different mechanisms in which DMN-CoA is either converted into the corresponding carnitine ester or hydrolyzed to 4,8-dimethylnonanoic acid plus CoASH. The latter pathway can only be operational if peroxisomes contain 4,8-dimethylnonanoyl-CoA thioesterase activity. In this paper we show that rat liver peroxisomes indeed contain 4,8-dimethylnonanoyl-CoA thioesterase activity. We have partially purified the enzyme involved from peroxisomes and identified the protein as the rat ortholog of a known human thioesterase using MALDI-TOF mass spectrometry in combination with the rat EST database. Heterologous expression studies in Escherichia coli established that the enzyme hydrolyzes not only DMN-CoA but also other branched-chain acyl-CoAs as well as straight-chain acyl-CoA-esters. Our data provide convincing evidence for the existence of the second pathway of acyl-CoA transport from peroxisomes to mitochondria by hydrolysis of the CoA-ester in peroxisomes followed by transport of the free acid to mitochondria, reactivation to its CoA-ester, and oxidation to CO(2) and H(2)O. (c)2002 Elsevier Science.     

Coupling historical prospection data and a remotely-sensed vegetation index for the preventative control of Desert locusts

Locusts are grasshopper species that exhibit phase polyphenism resulting in the expression of gregarious behaviors that favor the development of large devastating bands and swarms. Desert locust preventative management aims to prevent crop damage by controlling populations before they can reach high densities and form mass migrating swarms. The areas of potential gregarization for Desert locust are large and need to be physically assessed by survey teams for efficient preventative management. An ongoing challenge is to be able to guide where prospection surveys should occur depending on local meteorological and vegetation conditions. In this study, we analyzed the relationship between historical prospection data of Desert locust observations from 2005 to 2009 and spatio-temporal statistics of a vegetation index gathered by remote-sensing with the help of multiple models of logistic regression. The vegetation index was a composite Normalized Difference Vegetation Index (NDVI) given every 16 days and at 250 m spatial resolution (MOD13Q1 from MODIS satellite). The statistics extracted from this index were: (1) spatial means at different scales around the prospection point, (2) relative differences of NDVI variation through time before the prospection, and (3) large-scale summary of vegetation quantity. The multi-model framework showed that vegetation development a month and a half before the survey was amongst the best predictors of locust presence. Also, the local vegetation quantity was not enough to predict locust presence. Vegetation quantity on a scale of a few kilometers was a better predictor but varied non-linearly, reflecting specific biotope types that support Desert locust development. Using one of the best logistic regression models and NDVI data, we were able to derive a predictive model of probability of finding locusts in specific areas. This methodology should help in more efficiently focusing survey efforts on specific parts of the gregarization areas based on the predicted probability of locusts being present.     

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.     

Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA(Leu) (U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNA(Leu) (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.     

Relationship of embryogenic competence in maize (Zea mays L.) leaves to mitotic activity,cell cycle and nuclear DNA content

Axillary bud explants of 11 selected mature waratah clones were established in vitro on a modified Murashige & Skoog medium. Adequate proliferation of axillary shoots was achieved by optimisation of the growth regulator status of the culture medium. For the majority of clones, a three to six times rate of proliferation was achieved with 1.25 M BA and 1.0 M GA₃ without the occurrence of abnormalities. The white flowering clone did not respond favourably to the addition of GA₃ to the medium.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige & Skoog medium