Spatial and Temporal Gene Expression Differences in Core and Periinfarct Areas in Experimental Stroke: A Microarray Analysis

Background

A large number of genes are regulated to promote brain repair following stroke. The thorough analysis of this process can help identify new markers and develop therapeutic strategies. This study analyzes gene expression following experimental stroke.

Methodology/Principal Findings

A microarray study of gene expression in the core, periinfarct and contralateral cortex was performed in adult Sprague-Dawley rats (n = 60) after 24 hours (acute phase) or 3 days (delayed stage) of permanent middle cerebral artery (MCA) occlusion. Independent qRT-PCR validation (n = 12) was performed for 22 of the genes. Functional data were evaluated by Ingenuity Pathway Analysis. The number of genes differentially expressed was 2,612 (24 h) and 5,717 (3 d) in the core; and 3,505 (24 h) and 1,686 (3 d) in the periinfarct area (logFC>|1|; adjP<0.05). Expression of many neurovascular unit development genes was altered at 24 h and 3 d including HES2, OLIG2, LINGO1 and NOGO-A; chemokines like CXCL1 and CXCL12, stress-response genes like HIF-1A, and trophic factors like BDNF or BMP4. Nearly half of the detected genes (43%) had not been associated with stroke previously.

Conclusions

This comprehensive study of gene regulation in the core and periinfarct areas at different times following permanent MCA occlusion provides new data that can be helpful in translational research.     

Mexican Arnica Anti–Inflammatory Action: Plant Age is Correlated with the Concentration of Anti–Inflammatory Sesquiterpenes in the Medicinal Plant <Emphasis Type="Italic">Heterotheca inuloides</Emphasis> Cass. (Asteraceae)<Superscript>1</Superscript>

Mexican Arnica Anti–Inflammatory Action: Plant Age Is Correlated with the Concentration of Anti–inflammatory Sesquiterpenes in the Medicinal Plant Heterotheca inuloides Cass. (Asteraceae). Mexican árnica (Heterotheca inuloides Cass.) is a widely used anti–inflammatory medicinal plant in Mexican folk medicine. Although it has been suggested that plant age, fertilization, and harvesting regime influence the concentration of secondary compounds affecting the therapeutic activity of the plant, the effect of these variables on the concentration of the Mexican árnica anti–inflammatory compounds was not known. We quantified anti–inflammatory sesquiterpenes (caryolan–1, 9β–diol, cadalen–15–oic acid, 7–hydroxycadalene, 4–hydroxy–2–isopropyl–4, 7–dimethyl–1[4H] naftalinone, 7–hydroxy–4αH–3, 4–dihydrocadalene, β–caryophyllene, and β–caryophyllene epoxide) in Mexican árnica plants subjected to fertilization and successive harvests of flowering stems, conditions that mimic the cultivation and harvesting for árnica in México. Fertilization and successive harvesting and their interaction had no significant effect on the concentration of anti–inflammatory compounds. However, the concentrations of these compounds were 60% higher in flowering stems from 15–month–old plants than in those from 4– or 8–month–old plants and was independent of the number of harvests and fertilization regime applied.     

A first generation bovine BAC-based physical map

A first generation clone-based physical map for the bovine genome was constructed combining, fluorescent double digestion fingerprinting and sequence tagged site (STS) marker screening. The BAC clones were selected from an Inra BAC library (105 984 clones) and a part of the CHORI-240 BAC library (26 500 clones). The contigs were anchored using the screening information for a total of 1303 markers (451 microsatellites, 471 genes, 127 EST, and 254 BAC ends). The final map, which consists of 6615 contigs assembled from 100 923 clones, will be a valuable tool for genomic research in ruminants, including targeted marker production, positional cloning or targeted sequencing of regions of specific interest.     

The promises and problems of linkage analysis by using the current canine genome map

There have been major advances in the canine genome map over recent years, with an agreed karyotype for all 38 pairs of autosomes and an integrated linkage-radiation-hybrid map now available. An individual dog breed represents a closed, isolated population, and many suffer genetic diseases. Some are homologs of human conditions, and dogs represent naturally occurring, potentially useful large mammal models. Other conditions may be unique to this species, but study of these offers new insights into metabolic pathways. Linkage analysis may identify a locus linked to a particular condition. Dog pedigrees facilitate these studies by being relatively more informative than human families, although planned breeding strategies to maximize the informativeness may be required. A study of a late-onset, acquired heart disease, dilated cardiomyopathy (DCM), in Newfoundland dogs highlights some of the problems. The disease occurs naturally in pedigrees within the UK in dogs kept as pets and breeding stock. Problems encountered include confirmation of the mode of inheritance and determination of phenotype. A genome-wide linkage analysis study with over 200 markers failed to detect linkage. The study indicates that more detailed linkage maps are required, and further synteny information between dog and human gained before study of diseases affecting naturally occurring families of dogs from inbred pedigrees may have maximal chance of success in order to utilize this model.     

Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus)

Coat color genetics, when successfully adapted and applied to different mammalian species, provides a good demonstration of the powerful concept of comparative genetics. Using cross-species techniques, we have cloned, sequenced, and characterized equine melanocortin-1-receptor (MC1R) and agouti-signaling-protein (ASIP), and completed a partial sequence of tyrosinase-related protein 1 (TYRP1). The coding sequences and parts of the flanking regions of those genes were systematically analyzed in 40 horses and mutations typed in a total of 120 horses. Our panel represented 22 different horse breeds, including 11 different coat colors of Equus caballus. The comparison of a 1721-bp genomic fragment of MC1R among the 11 coat color phenotypes revealed no sequence difference apart from the known chestnut allele (C901T). In particular, no dominant black (E ^D) mutation was found. In a 4994-bp genomic fragment covering the three putative exons, two introns and parts of the 5′- and 3′-UTRs of ASIP, two intronic base substitutions (SNP-A845G and C2374A), a point mutation in the 3′-UTRs (A4734G), and an 11-bp deletion in exon 2 (ADEx2) were detected. The deletion was found to be homozygous and completely associated with horse recessive black coat color (A ^a /A ^a ) in 24 black horses out of 9 different breeds from our panel. The frameshift initiated by ADEx2 is believed to alter the regular coding sequence, acting as a loss-of-function ASIP mutation. In TYRP1 a base substitution was detected in exon 2 (C189T), causing a threonine to methionine change of yet unknown function, and an SNP (A1188G) was found in intron 2. Received: 22 November 2000 / Accepted: 07 February 2001     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Novel 12N-substituted matrinanes as potential anti-coxsackievirus agents

A series of novel 12N-substituted matrinane derivatives were synthesized and evaluated for their activities against coxsackievirus type B3 (CVB3) taking compound 1 as the lead. SAR analysis indicated that the introduction of a suitable heteroaromatic ring on the 12N-atom might be beneficial for the activity. Among them, compound 8a exhibited the highest potency against all CVB serotypes as well as CVA16 with IC₅₀ values ranging from 2.02 μM to 7.41 μM, indicating a broad-spectrum anti-coxsackieviruse effect. Furthermore, compound 8a demonstrated a good safety profile in vivo. Thus, we consider 12N-substituted matrinanes to be a promising family of anti-coxsackievirus agents, and compound 8a to be a promising drug candidate in the treatment of various diseases related to coxsackievirus infection.     

Glu–Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes

A Glu–Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization–mass (ESI?MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP–1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography–ESI?MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.     

Redefining the dose of the entomopathogenic fungus Metarhizium brunneum (Ascomycota,Hypocreales) to increase Fe bioavailability and promote plant growth in calcareous and sandy soils

Background and aims

Plants differ in their ability to use different nitrogen (N) chemical forms, these differences can be related to their ecology and drive community structure. The capacity to uptake intact organic N has been observed in plants of several ecosystems. However, soil organic N uptake by Mediterranean plants is unknown despite organic N being abundant in Mediterranean ecosystems. We compare the uptake of different N forms in two widespread coexisting Mediterranean forest trees with contrasting ecophysiological characteristics: Quercus ilex and Pinus halepensis.

Methods

To estimate root uptake rate of each N form we used equimolar solutions (1 mM N) of ¹⁵NO₃ ^?, ¹⁵NH₄ ⁺ and ¹⁵N-¹³C glycine.

Results

NH₄ ⁺ and glycine were taken up at a similar rate, but faster than NO₃ ^? in both species. Intact dual labeled glycine was found in both species, demonstrating that both species can absorb intact organic N.

Conclusions

Despite their ecological differences, both species had similar preference for N forms suggesting no fundamental niche complementarity for N uptake. The higher preference for NH₄ ⁺ and glycine over NO₃ ^? possibly reflects adaptation to the differing proportions of N forms in Mediterranean soils.

     

QTL mapping for traits associated with stress neuroendocrine reactivity in rats

In the present study we searched for quantitative trait loci (QTLs) that affect neuroendocrine stress responses in a 20-min restraint stress paradigm using Brown–Norway (BN) and Wistar–Kyoto–Hyperactive (WKHA) rats. These strains differed in their hypothalamic–pituitary–adrenal axis (plasma ACTH and corticosterone levels, thymus, and adrenal weights) and in their renin–angiotensin–aldosterone system reactivity (plasma renin activity, aldosterone concentration). We performed a whole-genome scan on a F₂ progeny derived from a WKHA × BN intercross, which led to the identification of several QTLs linked to plasma renin activity (Sr6, Sr8, Sr11, and Sr12 on chromosomes RNO2, 3, 19, and 8, respectively), plasma aldosterone concentration (Sr7 and Sr9 on RNO2 and 5, respectively), and thymus weight (Sr10, Sr13, and Srl4 on RNO5, 10, and 16, respectively). The type 1b angiotensin II receptor gene (Agtrlb) maps within the confidence intervals of QTLs on RNO2 linked to plasma renin activity (Sr6, highly significant; LOD = 5.0) and to plasma aldosterone level (Sr7, suggestive; LOD = 2.0). In vitro studies of angiotensin II–induced release of aldosterone by adrenal glomerulosa cells revealed a lower receptor potency (log EC₅₀ = −8.16 ± 0.11 M) and efficiency (E_max = 453.3 ± 25.9 pg/3 × 10⁴ cells/24 h) in BN than in WKHA (log EC₅₀ = −10.66 ± 0.18 M; E_max = 573.1 ± 15.3 pg/3 × 10⁴ cells/24 h). Moreover, differences in Agtr1b mRNA abundance and sequence reinforce the putative role of the Agtr1b gene in the differential plasma renin stress reactivity between the two rat strains.