Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

Background  

The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.     

Cardiopulmonary effects of butorphanol in sevoflurane‐anesthetized guineafowl (Numida meleagris)

ObjectiveTo evaluate the cardiopulmonary changes induced by intravenous butorphanol administration in guineafowl anesthetized with sevoflurane.Study designProspective experimental trial.AnimalsEight adult guineafowl (Numida meleagris) weighing 1.61 ± 0.49 kg were used for the study.MethodsBirds were anesthetized with sevoflurane and allowed to breathe spontaneously. After endotracheal intubation, end-tidal sevoflurane was adjusted to 1.0 individual sevoflurane MAC that was previously determined in triplicate using a standard bracketing technique. The brachial artery was catheterized for direct pressure measurement and blood sampling. Heart rate and rhythm were monitored by electrocardiography and respiratory rate was recorded. Baseline data were recorded 30 minutes after induction. Then, end-tidal sevoflurane was adjusted to 0.8 individual MAC and after 15 minutes physiologic variables were measured again. Subsequently, butorphanol (4 mg kg^?1) was administered intravenously over 10 seconds and physiologic responses were recorded at 1, 5, 10, 15, 20, 30 and 45 minutes after administration.ResultsButorphanol administration was associated with arrhythmias in all birds, including second-degree atrioventricular block, sinus arrest, ventricular and supraventricular tachycardia and ventricular premature complexes. Heart rate and arterial blood pressures decreased significantly 1 minute after butorphanol administration. Two birds developed severe hypotension, apnea and ventricular fibrillation 5 minutes after administration, and one died.Conclusions and clinical relevanceThe butorphanol dose (4 mg kg^?1) that produces clinically relevant sevoflurane MAC reduction in guineafowl caused severe adverse cardiopulmonary effects in two birds and was considered unsafe under the conditions used in this study.     

Assessment of copper and zinc status of farm horses and training thoroughbreds in south-east Queensland

The copper and zinc concentrations in the blood of stabled thoroughbred horses and in Australian Stock Horses mares at pasture, either late pregnant or lactating were determined by an atomic absorption spectroscopic method. The plasma concentration of the trace elements in these apparently normal horses were generally below the "normal" range. The plasma copper, caeruloplasmin copper, whole blood copper and plasma zinc concentrations in the stabled thoroughbreds were 0.76 +/- 0.19 micrograms/ml (n = 82), 0.56 +/- 0.14 micrograms/ml (n = 83), 0.75 +/- 0.18 micrograms/ml (n = 82) and 0.47 +/- 0.09 micrograms/ml (n = 83) respectively. The plasma copper and zinc concentrations of all the brood mares at pasture (pregnant and lactating) were 0.56 +/- 0.20 micrograms/ml and 0.47 +/- 0.11 micrograms/ml (n = 30). The plasma copper concentration of the pregnant group of mares (0.64 +/- 0.18 micrograms/ml; (n = 14) was greater than that of the lactating mares (0.49 +/- 0.21; (n = 16). Variation in the plasma copper concentration was also identified between stabled and farm horses, between horses of different stables and between horses of different ages. The proportion of plasma copper bound to caeruloplasmin was 73 +/- 11.8%. These low concentrations of copper and zinc in the plasma of apparently normal horses are of clinical significance since recent evidence has indicated that copper deficiency appears to promote the development of skeletal abnormalities in foals. An alternative to the use of a single plasma sample to identify the copper or zinc deficient horse was discussed.     

Two Methods of Vitrification Followed by In Vitro Culture of the Ovine Ovary: Evaluation of the Follicular Development and Ovarian Extracellular Matrix

The aim of this study was to evaluate the influence of two vitrification techniques on the extra cellular matrix (ECM) and ovarian follicular development. The ovarian cortex was fragmented (9 mm³) and divided into six groups, viz. fresh control, cultured control, vitrified by the Ovarian Tissue Cryosystem (OTC) method, conventional solid surface vitrification (SSV) method, OTC/cultured and SSV/cultured. Follicles from all the fragments were analysed for morphology, development and viability. The ECM was evaluated based on the condition of collagen and reticular fibres and the immunolocalization of type I collagen and fibronectin. After 7 days of culture, the tissue vitrified by OTC revealed a higher percentage (p < 0.05) of morphologically normal (30.66%) and viable (60.00%) follicles when compared with those vitrified using the SSV technique (21.33% and 23.00%). In all the fragments cultured, regardless of the vitrification method, a significantly higher percentage of developing follicles was observed when compared with the non‐cultured tissue. Analysis of the type I collagen showed increased immunostaining after the in vitro culture in the vitrified fragments. In conclusion, the OTC is better for preserving the follicular viability and morphology and maintaining the integrity of the extracellular matrix components of the ovine ovary.     

Pyrrolizidine alkaloidosis of cattle associated with Senecio lautu

Summary Serious incidents of pyrrolizidine alkaloidosis of cattle in 10 herds exposed to the Australian native plant, Senecio lautus (Asteraceae), were seen in central Queensland during 1988–1992. The deaths of 226 cattle were recorded. A mean of 8% of cattle died in affected groups (range 2 to 58%). Sickness and deaths usually occurred some months after access to S lautus. Typically, affected cattle lost body condition to the point of emaciation before dying and had persistent diarrhoea. Some animals developed abnormal behaviour and died after a shorter illness. Liver specimens from affected cattle in all herds contained lesions consistent with pyrrolizidine alkaloidosis. Thin layer chromatography of extracts of blood and liver samples from cattle from 5 herds detected pyrrolic metabolites. The identity of these was confirmed by mass spectroscopy on samples from one herd. Unseasonal autumn and winter rain after a dry summer appeared to favour growth of S lautus at the expense of other pasture species. A subsequent dry period promoted consumption of S lautus and was followed by a cluster of poisoning incidents.     

Cyclooxygenases 1 and 2 inhibition and analgesic efficacy of dipyrone at different doses or meloxicam in cats after ovariohysterectomy

ObjectiveTo evaluate the cyclooxygenases (COX) inhibition, adverse effects and analgesic efficacy of dipyrone or meloxicam in cats undergoing elective ovariohysterectomy.Study designProspective, blinded, randomized, clinical study.AnimalsA total of 30 healthy young cats.MethodsThe cats were randomly assigned to three postoperative groups: D25 (dipyrone 25 mg kg^?1 every 24 hours), D12.5 (dipyrone 12.5 mg kg^?1 every 12 hours) and M (meloxicam 0.1 mg kg^?1 every 24 hours). In the first 24 hours, the drugs were administered intravenously (IV), and then orally for 6 (dipyrone) or 3 days (meloxicam). Prostanoids thromboxane B₂ and prostaglandin E₂ concentrations served as indicators of COX activity and, with physiological variables and pain and sedation scores, were measured for 24 hours after first analgesic administration. Rescue analgesia (tramadol, 2 mg kg^?1 IV) was provided if Glasgow feline composite measure pain scale (CMPS-Feline) ≥5. Laboratory tests included symmetric dimethylarginine and adverse effects were evaluated regularly up to 7 and 10 days after surgery, respectively. Parametric and nonparametric data were analyzed with two-way anova and Kruskal-Wallis tests, respectively (p < 0.05).ResultsIn the first half hour after analgesic administration, COX-1 activity was close to zero and remained significantly lower than before drug administration for 24 hours in all groups. The inhibition of COX-2 activity was significant for 30 minutes in all groups and up to 4 hours in group M. No alterations in laboratory tests or significant adverse effects were observed. Pain scores and need for rescue analgesia did not differ statistically among groups.ConclusionsDipyrone at both doses and meloxicam provided a nonselective inhibition of COX-1 and -2 activities and effective analgesia without causing significant adverse effects or laboratory tests alterations.Clinical relevanceDipyrone at both doses provides equally effective analgesia without causing adverse effects in cats undergoing ovariohysterectomy.     

Effect of selenium supplementation on semen characteristics of Brazil's ram

The aim of this study was to investigate the effects of different concentrations of oral supplementation with selenium (Se) upon ram sperm parameters. Thirty rams managed in stall under intensive system were used and divided into five groups (six animals per group) as follows: control group (G1) mineral mixture supplementation without Se, group 2 (G2) mineral mixture supplemented with 5 mg/kg Se, group 3 (G3) supplemented with 10 mg/kg Se, group 4 (G4) supplemented with 15 mg/kg Se and group 5 (G5) supplemented with 20 mg/kg Se. For each group, there was an adjustment period of 14 days. The experimental period was 350 days. Every 56 days, the animals were weighed and semen samples were collected by electroejaculation. Semen analysis included volume, mass moviment, total motility, vigour, concentration and morphology. For plasmatic and acrosomal membrane integrity evaluation and mitochondrial membrane potential were used a combination of fluorescent probes. Differences between means values obtained by analysis of variance were verified by Tukey test with 5% probability. There was no statistical difference between treatment groups in relation to volume, mass moviment, total motility, vigour, concentration, plasma and acrosomal membrane integrity (p > .05). Sperm morphology was different between treatment groups, the G1 (0 mg of selenium) had the highest percentage of major defects (11.11 ± 1.11^a; p < .05). It was concluded that selenium decrease the percentage of sperm defects and did not directly influence on ram sperm volume, mass moviment, total motility, vigour, concentration and membrane integrity.