Metabolic engineering of tomato fruit organic acid content guided by biochemical analysis of an introgression line.

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.

Feline cholangitis: a necropsy study of 44 cats (1986-2008).

Forty-four cats diagnosed with moderate to severe cholangitis at necropsy are described. The population comprised 0.86% of all feline necropsies performed during the 22-year study period. Liver specimens were classified as acute neutrophilic cholangitis (ANC), chronic neutrophilic cholangitis (CNC), lymphocytic cholangitis (LC) or chronic cholangitis associated with liver fluke infestation (CC) based on the World Small Animal Veterinary Association (WSAVA) classification scheme. ANC (seven) and CNC (33) comprised the majority of cases. In contrast to previous descriptions, overlap was seen in clinical findings between ANC and CNC subtypes. Results suggest that liver enzyme activity may not predict degree of inflammation. Severity of inflammation varied between liver sections in individual cats, underscoring the need to obtain biopsy samples from multiple sites. Inflammatory bowel disease (50%), pancreatitis (60%), or both (32%) commonly accompanied cholagitis. We conclude that cholangitis is not a common cause of feline mortality. Most cats that succumb to cholangitis have ANC or CNC, and concurrent disease contributes to death in many.

Use of a self-expanding metallic stent for the treatment of a urethral stricture in a young cat.

A 4-month-old intact male domestic shorthair cat was evaluated for urinary outflow obstruction after several weeks of medical management for traumatic urethral rupture. Positive-contrast retrograde urethrography and anterograde cystoscopy performed 4 weeks after the initial urethral injury confirmed a stricture approximately 1cm distal to the bladder trigone at the site of the initial urethral tear. A self-expanding metallic urethral stent (SEMS) was placed under fluoroscopic guidance to relieve the urethral stricture and re-establish luminal patency. After stent placement, the cat was able to void urine normally with minimal urinary incontinence noted. This resolved several months post-stent placement. No known clinical complications persisted other than mild intermittent hematuria.

Clinical peripheral neuropathy associated with diabetes mellitus in 3 dogs.

Clinical and electrodiagnostic findings in 3 spontaneously diabetic dogs with clinical peripheral neuropathy (PN) are reported. Clinical signs of a PN may develop in diabetic dogs with adequate glycemic control. In addition, laryngeal paralysis may develop in association with diabetes mellitus in dogs with clinical PN.

Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis.

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO2 output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.

Matrix isolation and density functional theory study of bis(trifluoromethyl)dioxodiazine: a photodimer of trifluoronitrosomethane.

Trifluoronitrosomethane (CF3NO) was trapped in rare gas matrixes and irradiated at 633 and 670 nm. The infrared spectra of the postirradiation samples exhibit features consistent with cis and trans conformers of bis(trifluoromethyl)dioxodiazine, a previously uncharacterized species. The concentration dependence of the formation of the dimer is consistent with a mechanism in which monomers trapped in adjacent sites undergo excitation and subsequent reaction. The dimers reversibly form the monomer when irradiated with ultraviolet light. Density functional theory was used to determine the structure of the dimers and predict their infrared and Raman spectra. The predicted vibrational frequencies are in agreement with those observed. A third (skewed) conformation was predicted to have a triplet ground state, but no evidence of this species was observed. All three dimers exhibit significant diradical character, as evidenced by comparatively low N-N and high N-O stretching frequencies. Transition-state calculations predict the dimerization barrier to range from 17.1 (cis) to 35.0 (trans) kJ mol(-1) for the singlet dimers and to be 62.1 kJ mol(-1) for the triplet dimer. This is an example of nitroso dimerization that requires electronic excitation to proceed.