Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins

The fungal disease Fusarium head blight occurs on wheat (Triticum spp.) and barley (Hordeum vulgare L.) and is one of the worldwide problems of agriculture. It can be caused by various Fusarium species. We are characterizing the proteinases of F. culmorum to investigate how they may help the fungus to attack the grain. A trypsin-like proteinase has been purified from a gluten-containing culture medium of F. culmorum. The enzyme was maximally active at about pH 9 and 45 degrees C, but was not stable under those conditions. It was stabilized by calcium ions and by the presence of other proteins. The proteinase was most stable at pH 6-7 at ambient temperatures, but was quickly inactivated at 50 degrees C. It was strongly inhibited by p-amidino phenylmethylsulfonyl fluoride (p-APMSF), and soybean trypsin and Bowman-Birk inhibitors, and it preferentially hydrolyzed the peptide bonds of the protein substrate beta-purothionin on the C-terminal side of Arg (mainly) and Lys residues. These characteristics show that it is a trypsin-like proteinase. In addition, its N-terminal amino acid sequence was 88% identical to that of the F. oxysporum trypsin-like enzyme. The proteinase hydrolyzed the D hordein and some of the C hordeins (the barley storage proteins). This enzyme, and a subtilisin-like proteinase that we recently purified from the same organism, possibly play roles in helping the fungus to colonize grains.     

A comparison of extradural and intravenous methadone on intraoperative isoflurane and postoperative analgesia requirements in dogs

Objective To compare the effects of intravenous (IV) and extradural (ED) methadone on end‐tidal isoflurane concentration (Fe ′ISO) and postoperative analgesic requirements in dogs undergoing femoro‐tibial joint surgery. Study Design Randomized, blinded, clinical study. Animals Twenty‐four healthy client‐owned dogs undergoing surgical repair of ruptured cruciate ligaments. Methods Dogs were randomly assigned to two groups of 12 animals and received either ED or IV methadone (0.3 mg kg^?1 diluted with saline to 0.2 mL kg^?1). Pre‐anaesthetic medication was IV acepromazine (0.05 mg kg^?1). Anaesthesia was induced with propofol and maintained initially with an Fe ′ISO of 1.0% delivered in oxygen. Methadone was injected with the dogs in sternal recumbency; the observer was unaware of the administration route. At 10 minutes (stimulation 1) and 20 minutes (stimulation 2) after methadone administration pelvic limb reflexes were tested by digit‐clamping. The time at skin incision (stimulation 3), joint‐capsule incision (stimulation 4), tibial tuberosity drilling (stimulation 5), fabellar suturing (stimulation 6) and extracapsular tightening (stimulation 7) were noted. Changes in heart rate (HR) and respiratory rate and arterial blood pressure associated with surgery were recorded along with the corresponding Fe ′ISO. After 20 minutes of anaesthesia, Fe ′ISO was decreased to the minimum required to maintain stable anaesthesia. Immediately after tracheal extubation, 1, 2, 3 and 6 hours postoperatively and on the morning after surgery, the degree of pain present was assessed using a numerical rating scale. The HR, respiratory rates and blood pressure were also recorded at these times. Serum cortisol and blood glucose concentrations were measured before pre‐anaesthetic medication and at each postoperative pain scoring interval except at 1 and 2 hours. Ketoprofen (2 mg kg^?1), carprofen (4 mg kg^?1) or meloxicam (0.2 mg kg^?1) were given by subcutaneous injection whenever pain scoring indicated moderate discomfort was present. Results Controlled ventilation was required in six dogs which stopped breathing after IV methadone. The median Fe ′ISO at stimulus 5 was 1.0% in the IV and 0.83% in the ED group. At stimulus 6, Fe ′ISO was 1.0% in the IV and 0.8% in the ED group; the difference was statistically significant (p ≤ 0.05). There was no significant difference in the duration of postoperative analgesia associated with administration route. Conclusions Extradural methadone significantly reduces the isoflurane requirement compared with IV methadone during femoro‐tibial joint surgery in dogs. Clinical relevance Extradural methadone provides safe and effective pain relief in dogs undergoing cruciate ligament repair.