Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.     

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.     

Expression of a mitochondrial peroxiredoxin prevents programmed cell death in Leishmania donovani

Leishmania promastigote cells transmitted by the insect vector get phagocytosed by macrophages and convert into the amastigote form. During development and transformation, the parasites are exposed to various concentrations of reactive oxygen species, which can induce programmed cell death (PCD). We show that a mitochondrial peroxiredoxin (LdmPrx) protects Leishmania donovani from PCD. Whereas this peroxiredoxin is restricted to the kinetoplast area in promastigotes, it covers the entire mitochondrion in amastigotes, accompanied by dramatically increased expression. A similar change in the expression pattern was observed during the growth of Leishmania from the early to the late logarithmic phase. Recombinant LdmPrx shows typical peroxiredoxin-like enzyme activity. It is able to detoxify organic and inorganic peroxides and prevents DNA from hydroxyl radical-induced damage. Most notably, Leishmania parasites overexpressing this peroxiredoxin are protected from hydrogen peroxide-induced PCD. This protection is also seen in promastigotes grown to the late logarithmic phase, also characterized by high expression of this peroxiredoxin. Apparently, the physiological role of this peroxiredoxin is stabilization of the mitochondrial membrane potential and, as a consequence, inhibition of PCD through removal of peroxides.     

Inflammatory macrophages induce Nrf2 transcription factor-dependent proteasome activity in colonic NCM460 cells and thereby confer anti-apoptotic protection

Adaptation of epithelial cells to persistent oxidative stress plays an important role in inflammation-associated carcinogenesis. This adaptation process involves activation of Nrf2 (nuclear factor-E2-related factor-2), which has been recently shown to contribute to carcinogenesis through the induction of proteasomal gene expression and proteasome activity. To verify this possible link between inflammation, oxidative stress, and Nrf2-dependent proteasome activation, we explored the impact of inflammatory (M1) macrophages on the human colon epithelial cell line NCM460. Transwell cocultures with macrophages differentiated from granulocyte monocyte-colony-stimulating factor-treated monocytes led to an increased activity of Nrf2 in NCM460 cells along with an elevated proteasome activity. This higher proteasome activity resulted from Nrf2-dependent induction of proteasomal gene expression, as shown for the 19 and 20 S subunit proteins S5a and α5, respectively. These effects of macrophage coculture were preceded by an increase of reactive oxygen species in cocultured NCM460 cells and could be blocked by catalase or by the reactive oxygen species scavenger Tiron, whereas transient treatment of NCM460 cells with H(2)O(2) similarly led to Nrf2-dependent proteasome activation. Through the Nrf2-dependent increase of proteasomal gene expression and proteasome activity, the sensitivity of NCM460 cells to tumor necrosis factor-related apoptosis-inducing ligand- or irinotecan-induced apoptosis declined. These findings indicate that inflammatory conditions such as the presence of M1 macrophages and the resulting oxidative stress are involved in the Nrf2-dependent gain of proteasome activity in epithelial cells, e.g. colonocytes, giving rise of greater resistance to apoptosis. This mechanism might contribute to inflammation-associated carcinogenesis, e.g. of the colon.     

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model

Abstract

Context: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals.

Objective: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice.

Methods: Mice were whole body irradiated with X-rays (2?Gy–15?Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature.

Results: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates.

Conclusion: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.     

Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

Endogenous synthesis of n-3 polyunsaturated fatty acids in Fat-1 mice is associated with increased mammary gland and liver syndecan-1

Long chain n-3 PUFA have been shown to have chemopreventive properties against breast cancer through various mechanisms. One pathway, studied in human breast cancer cell lines, involves upregulation of the proteoglycan, syndecan-1 (SDC-1) by n-3 PUFA-enriched LDL. Using Fat-1 mice that are able to convert n-6 to n-3 PUFA, we tested whether SDC-1 level in vivo is elevated in mammary glands due to endogenously synthesized rather than LDL-derived n-3 PUFA. Female Fat-1 and wild type (wt) mice were fed an n-6 PUFA- enriched diet for 7 weeks. Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively). Lipoprotein percent n-3 PUFA was also similar between groups. However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice. This was accompanied by higher SDC-1 in mammary glands and livers of Fat-1 mice, thus demonstrating that endogenously synthesized n-3 PUFA may upregulate SDC-1 in the presence of high dietary n-6 PUFA.