N form–dependent growth retardation of Arabidopsis thaliana seedlings as revealed from physiological and microarray studies

Ammonium (NH ) nutrition causes retardation of growth in many plant species. In Arabidopsis grown with NH as the sole N source, growth retardation occurs already at early stages before photosynthesis has come to its full power. In order to describe the peculiarities of these retarded plants, they were compared with nitrate (NO )‐grown plants of the same age of 15 d. Photosynthetic activity as measured by CO₂ uptake per unit chlorophyll is half as high in NH ‐grown seedlings as in NO ‐grown ones. This finding is confirmed by the analysis of chlorophyll fluorescence. Chloroplasts of NO ‐grown, but not of NH ‐grown, seedlings show starch deposits after 5 h of illumination with 40 μmol m^–2 s^–1. Gene‐expression analysis based on cDNA microarray and on Northern blots provide a clue about the biochemical background. After the first 2 weeks of growth, it seems that NO ‐grown seedlings subsist mainly on normal photosynthesis, whereas NH ‐grown seedlings still use lipids from the seeds stored in oleosomes. Corresponding to this observation, the mRNAs for enzymes of β‐oxidation are more strongly expressed in NH ‐grown seedlings. Different carbohydrate sources for sucrose synthesis are indicated by different gene expression. Higher gene expression of fructose bisphosphate aldolase (cytosolic isoform) in NO ‐grown seedlings indicates the dependence on photosynthesis, whereas a higher gene expression of PEP carboxykinase in NH ‐grown seedlings points to a prominent role of β‐oxidation of storage lipids still present.     

Differential effects of domoic acid and E. coli lipopolysaccharide on tumor necrosis factor-alpha, transforming growth factor-beta1 and matrix metalloproteinase-9 release by rat neonatal microglia: evaluation of the direct activation hypothesis

The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).     

Relevance of isotopic and molecular biomarkers for the authentication of milk according to production zone and type of feeding of the cow

The first objective of the present paper was to assess the potential of both isotopic ( (18)O/ (16)O in milk water) and molecular biomarkers (terpenes, fatty acids, carotenoids, and vitamins) and milk color to discriminate the production zone (lowland or upland areas) from which 49 tanker bulk milks were collected over one year from a total of 204 farms. The milk water (18)O enrichment was higher in lowland (<500 m altitude) than in upland (>700 m altitude), but the delta (18)O values failed to discriminate systematically the production zone at the scale of the year because of its high variability related to the sampling period. In contrast with vitamins A and E, carotenoids, and milk color measurements, terpenes and fatty acids were confirmed to be relevant tracers of the production zone. The milk compounds with the strongest discriminative potential were fatty acids, which were determined by high-resolution gas chromatography. The calculation of fatty acid ratios, which permits the limitation of using fatty acid relative quantity expressed in percentage of total fatty acids to be overcome, was shown to be particularly relevant in discriminating upland from lowland milk ratios. The selection of two pairs of ratios, namely, iso-C17:0/C18:3 n-3 and iso-C15:0/iso-C14:0, enabled the authentication of 100% of the highland versus lowland milks whatever the season. The second objective was to evaluate the relevance of fatty acid composition to discriminate milks according to the proportion of corn silage in the diets of dairy cows. The selection of two fatty acids ratios, namely, trans11 cis15-C18:2/trans11-C18:1 and cis9-C16:1/iso-C16:0, enabled the correct classification of 100% of the milk samples according to the proportion of corn silage in the basic fodder rations (<25% vs >30%). The relationship between the milk production zone and the type of forage fed to the cows is discussed.     

Assessment of the use of RNA quality metrics for the screening of articular cartilage specimens from clinically normal dogs and dogs with osteoarthritis

OBJECTIVE: To assess 2 methods of RNA purification by use of different quality metrics and identify the most useful metric for quality assessment of RNA extracted from articular cartilage from dogs with osteoarthritis. SAMPLE POPULATION: 40 articular cartilage specimens from the femoral heads of 3 clinically normal dogs and 37 dogs with osteoarthritis. PROCEDURES: RNA was extracted from articular cartilage by 2 purification methods. Quality metrics of each sample were determined and recorded by use of a UV spectrophotometer (Spec I; to determine the 260 to 280 nm absorbance ratio [A(260):A(280) ratio]), a second UV spectrophotometer (Spec II; to determine A(260):A(280) and A(260):A(230) absorbance ratios), and a microfluidic capillary electrophoresis analyzer (to determine the ribosomal peak ratio [RR], degradation factor [DF], and RNA integrity number [RIN]). The RNA was extracted from affected (osteoarthritic) articular cartilage and assessed with the same quality metrics. Metric results were compared with visual analysis of the electropherogram to determine the most useful RNA quality metric. RESULTS: No differences in methods of RNA purification were determined by use of quality metrics. The RNA extracted from unaffected (normal) cartilage was of higher quality than that extracted from affected (osteoarthritic) cartilage, as determined by the RIN and Spec II A(260):A(230) ratio. The RIN and RR were the most sensitive metrics for determining RNA quality, whereas the DF was most specific. A significant proportion (32%) of RNA extracted from osteoarthritic articular cartilage specimens was determined as being of low quality. CONCLUSIONS AND CLINICAL RELEVANCE: No single metric provided a completely sensitive and specific assessment of the quality of RNA recovered from articular cartilage.     

A review of the small canine piroplasms from California: Babesia conradae in the literature

Small piroplasms as a cause of canine babesiosis in southern California were first documented in 1990. Initially these piroplasms were considered to be Babesia gibsoni, the only small Babesia parasite known to infect dogs at that time. In the following decade, the use of molecular analysis made it clear that small canine Babesia in fact are comprised of at least three distinct species, and the isolates from dogs in southern California were not B. gibsoni. Molecular, antigenic, and morphological characteristics of the southern California species of canine piroplasm supported naming it as a distinct species, Babesia conradae. The renaming of this species prompted this literature review of small canine piroplasms in California in order to clarify clinical, diagnostic, epidemiological, and molecular characteristics of B. conradae in comparison to other small canine piroplasms. Clinical symptoms of B. conradae are similar to those of B. gibsoni; however, B. conradae infections may be more pathogenic, resulting in higher parasitaemia and more pronounced anaemia when compared with B. gibsoni-infected dogs. The immunofluorescent antibody test is the most commonly used test to diagnose B. conradae. It is important to specify which small Babesia species to test for since there is little serological cross reactivity between the small canine Babesia antigens or cross-detection in the newer molecular tests. Molecular characterization of B. conradae, based principally on the 18S small subunit rRNA gene, and recently the second internal transcribed spacer region, demonstrate that B. conradae is most closely related to piroplasms recovered from humans and animals in the western United States.     

Diagnosis of mediastinal masses in dogs by flow cytometry

BACKGROUND: Biopsy of mediastinal masses can be invasive, but the procedure may be necessary if cytology of a mass aspirate is inconclusive. The 2 most common mediastinal masses, lymphoma and thymoma, may both be comprised of small lymphocytes. We investigated the ability of flow cytometry to distinguish between these 2 neoplasms. HYPOTHESIS: Flow cytometry of mediastinal mass aspirates may provide a definitive diagnosis of thymoma or lymphoma, reducing the need for biopsy. ANIMALS: Dogs with mediastinal masses presenting to the Veterinary Teaching Hospital/Animal Cancer Center were included in the study. METHODS: Aspirates obtained over 2 years that met the inclusion criteria (i.e. sufficient viable cells and a definitive diagnosis by means other than flow cytometry) were analyzed by flow cytometry to determine the percentage of cells expressing B- and T-cell markers, and co-expressing CD4 and CD8. RESULTS: All cases of thymoma (n = 6) consisted of > or = 10% lymphocytes coexpressing CD4 and CD8, a phenotype that is characteristic of thymocytes, whereas 6 of 7 lymphomas contained <2% CD4+CD8+ lymphocytes. The CD4+CD8+ lymphoma could be readily distinguished flow cytometrically from thymoma by light scatter properties. The phenotypes of the remaining lymphomas were CD4+ T cell (4), CD34+ (1) and B cell (1). CONCLUSIONS: Our studies demonstrate that flow cytometry is a useful tool for discriminating mediastinal masses. Lymphocyte-rich mediastinal masses could be unambiguously identified by flow cytometry in 13/13 cases.     

Reported antibiotic use in 90 swine farms in Alberta

Antibiotic use was described using a convenience sample of 90 Alberta swine farms representing approximately 25% of the Alberta market swine production. Data on the use of antibiotics were collected through an on-farm interview questionnaire. The vast majority of antibiotics were used in feed. The chlortetracycline/sulfamethazine/penicillin combination and tylosin were the most frequently used in-feed antibiotics in weaners and growers/finishers, respectively. The use of antibiotics through water was reported mostly occasionally in all categories. The use of injectable antibiotics was reported mostly in sick pigs. Penicillin was the most common in-water and injectable antibiotic in all categories. The apparent low frequency of critically important antimicrobials for use in humans (quinolones and 3rd generation cephalosporins) is an encouraging finding from a public health perspective. The widespread and frequently reported use of penicillin and tetracycline are of public health concern considering that both antimicrobials are also used for therapeutic purposes in human medicine.