CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.     

Sex, secondary compounds and asymmetry. Effects on plant–herbivore interaction in a dioecious shrub

We analysed the links between herbivory, anthraquinone content and developmental instability of leaves in Rhamnus alpinus, taking into account possible effects of sexual dimorphism. The amount of leaf loss caused by herbivores averaged 3%, rarely exceeding 25%. Leaf losses were evenly distributed in the shrubs, with highest variability among leaves of the same shoot, thus hiding possible shrub, sex or population effects. This pattern of herbivory implies a shifting of caterpillars from one leaf to another before consuming all readily available material. We suggest that this behaviour might be triggered by a short-term change in leaf palatability by means of an increase in the production of secondary compounds. Supporting this hypothesis, we have found a higher anthraquinone content in damaged leaves compared with undamaged ones. The leaves of male plants exhibited a higher concentration of anthraquinones than those of females, which contrasts with classic hypotheses. We relate this to the lower rate of biomass increase in males, which should allow them to allocate more resources to defence. Leaves showed fluctuating asymmetry (FA), but we did not find any relationship between the degree of asymmetry and sex, herbivory or anthraquinone content at any level considered. Therefore, FA cannot be considered as an indicator of susceptibility to damage by herbivores or of the ability to induce the production of defensive compounds in R. alpinus.     

T-Kininogen Inhibits Fibroblast Proliferation in the G1 Phase of the Cell Cycle

By using synthetic protease inhibitors, several investigators have demonstrated that cysteine proteinases are required for cell proliferation. Kininogens are potent and specific physiological inhibitors of cysteine proteinases. We have used several mouse fibroblast-derived cell lines that express biologically active T-kininogen under the control of the mouse metallothionein promoter to test its effect on cell proliferation. Our results indicate that expression of T-kininogen results in diminished proliferative capacity, as measured by reduced cell numbers, both in logarithmically growing cultures and in G₀ cells induced to proliferate in response to serum. Furthermore, both fluorescence-activated cell sorting (FACS) analysis and incorporation of radioactive precursors into DNA suggest that the cells are unable to progress from G₀ through the S phase of the cell cycle in response to serum stimulation. However, we find that T-kininogen-expressing cell lines are still capable of responding to growth factors present in the serum, both by activating the ERK pathway and by expressing early genes, such as c-Fos and c-Jun. Thus, our results suggest that inhibition of cysteine proteinases by T-kininogen leads to inhibition of cell proliferation between the G₁ and S phases of the cell cycle.     

Mineral nutrition and growth of tropical maize as affected by soil acidity

Soil constraints linked to low pH reduce grain yield in about 10% of the maize growing area in tropical developing countries. The aim of this research was to elucidate the reasons for this maize yield reduction on an oxisol of Guadeloupe. The field experiment had two treatments: the native non-limed soil (NLI, pH 4.5, 2.1 cmol Al kg^–1, corresponding to 20% Al saturation), and the same soil limed 6 years prior to the experiment (LI, pH 5.3, 0 cmol Al kg^–1). The soils were fertilized with P and N. The above-ground biomass, root biomass at flowering, grain yield and yield components, leaf area index (LAI), light interception, radiation-use-efficiency (RUE), P and N uptake, soil water storage, and soil mineral N were measured during the maize cycle. The allometric relationships between shoot N concentration, LAI and above-ground biomass in LI were similar to those reported for maize cropped in temperate regions, indicating that these relationships are also useful to describe maize growth on tropical soils without Al toxicity. In NLI, soil acidity severely affected leaf appearance, leaf size and consequently the LAI, which was reduced by 60% at flowering, although the RUE was not affected. Therefore, the reduction in the above-ground biomass (30% at flowering) and grain yield (47%) were due to the lower LAI and light interception. At flowering, the root/shoot ratio was 0.25 in NLI and 0.17 in LI, and the root biomass in NLI was reduced by 64% compared to LI. Nitrogen uptake was also reduced in NLI in spite of high soil N availability. Nevertheless, shoot N concentration vs aboveground biomass showed a typical decline in both treatments. In NLI, the shoot P concentration vs above-ground biomass relationship showed an increase in the early stages, indicating that P uptake and root-shoot competition for the absorbed P in the early plant stages controlled the establishment and the development of the leaf area.     

Caracterización morfológica y molecular del antagonismo entre el endofito Diaporthe sp. aislado de frailejón (Espeletia sp.) y el fitopatógeno Phytophthora infestans

Endophytic fungi produce a great variety of secondary metabolites both in vivo and in vitro. In this study, we characterized the ability of a sterile-mycelium endophytic fungus isolated from Espeletia sp. to control the growth of Phytophthora infestans in Petri dishes. Sequence from the ITS regions (internal transcribed spacer) of the endophyte showed 94% similarity to Diaporthe phaseolorum's. The antagonistic interaction between Diaporthe sp. and P. infestans was evaluated in three different culture media. Diaporthe sp. showed an antagonistic effect towards P. infestans, with some variation depending on which medium was used. In an attempt to identify possible genes involved in this antagonism, we detected a gene from the endophyte encoding an amylase, which was differentially expressed during this biotic interaction.     

Serum T-kininogen levels increase two to four months before death.

We have reported an accumulation of T-kininogen mRNA in the liver of aging Sprague-Dawley rats. T-kininogen is a cysteine proteinase inhibitor. Since a disruption of the intracellular protein degradation machinery is known to occur during senescence, we wished to further define the role of this protein in the aging process. As a first step, we have measured T-kininogen levels both in serum and within the liver. We have found that serum protein levels are indeed augmented during senescence, although not as dramatically as the mRNA (2.5-fold versus 8.3-fold). Immunocytochemistry, as well as Western blot analysis suggests that this is due to the presence of T-kininogen within hepatic cells in aged rats. Life-long dietary restriction, a known age-prolonging treatment, decreases the overexpression of the protein in 24-month-old rats. Later, diet-restricted animals still show an increased expression from the gene, the effect being delayed but not abolished by dietary manipulation. Interestingly, a longitudinal study indicated the existence of a positive correlation between the time of increase of serum T-kininogen and the time of death of the animal. Serum T-kininogen was found to increase 2.5-4 months before death.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.