Multicolor and electron microscopic imaging of connexin trafficking

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.     

Preliminary study of plasma vascular endothelial growth factor (VEGF) during low- and high-dose radiation therapy of dogs with spontaneous tumors

High plasma vascular endothelial growth factor (VEGF) concentrations are associated with radiation resistance and poor prognosis. After an exposure to ionizing radiation in cell culture an early phase and a late phase of increased VEGF have been documented. The activation was dependent on the radiation dose. Therefore, the purpose of this study was to measure baseline plasma VEGF and changes in VEGF over the course of fractionated radiation therapy in dogs with spontaneous tumors. Dogs with tumors had a significantly higher pretreatment plasma VEGF than did dogs without tumors. Immediately after irradiation no increased plasma VEGF was observed. Over the course of radiation therapy there was an increased plasma VEGF in dogs treated with low doses per fraction/high total dose, whereas plasma VEGF remained stable in dogs irradiated with high doses per fraction/low total dose. The regulatory mechanisms are very complex, and therefore the value of plasma VEGF measurements as an indirect marker of angiogenesis induced by radiotherapy is limited.     

The effect of epidural morphine on the minimum alveolar concentration of isoflurane in cats

This study was undertaken to evaluate the effect of 3 different doses of epidurally administered morphine sulphate on the minimum alveolar concentration (MAC) of isoflurane in healthy cats. Five 4-year-old, spayed female cats weighing 4.7 ± 0.8 kg were allocated randomly to receive one of 3 doses of morphine on each study day. The 3 doses of morphine were 0.05, 0.1 and 0.2 mg/kg bwt and each cat was studied 3 times so that each cat received all doses. On each study day, cats were anaesthetised with isoflurane and instrumented. The MAC of isoflurane was determined in triplicate and morphine sulphate was administered via an epidural catheter chronically implanted prior to the study. Maximum MAC reduction was determined over the following 2 h. At the end of the study cats were allowed to recover. There was a significant reduction in MAC of isoflurane, with all doses of epidural morphine (P<0.05). The maximum reduction in MAC of isoflurane after 0.05 mg/kg bwt, 0.10 mg/kg bwt and 0.20 mg/kg bwt morphine was 21.4 ± 9.796, 30.8 ± 9.696, and 30.2 ± 6.8%, respectively, with no significant difference between doses. Systolic, mean and diastolic blood pressure, heart rate, respiratory rate and arterial pH decreased significantly whereas arterial carbon dioxide tension increased significantly after morphine administration (P<0.05). The means for all variables returned to pre-morphine values when the end-tidal isoflurane concentration was reduced to the new MAC point. In conclusion, epidural morphine decreased the concentration of isoflurane required to prevent movement in response to noxious mechanical stimulation to the tail base. A similar effect may be seen clinically allowing lower doses of isoflurane to be used to provide surgical anaesthesia for procedures involving the hind limbs, pelvis and tail.     

Resistance to Botrytis cinerea in Solanum lycopersicoides is Dominant in Hybrids with Tomato, and Involves induced Hyphal Death

Botrytis cinerea causes gray mold disease and affects hundreds of plant species, including tomato (Lycopersicon esculentum). The wild nightshade, Solanum lycopersicoides, is cross compatible with tomato and is more resistant to B. cinerea, thus representing a potential source for crop improvement. Tests involving droplet inoculation of detached leaves and spray inoculation of entire seedlings demonstrated that resistance to B. cinerea varies among S. lycopersicoides accessions, with S. lycopersicoides LA2951 being the most resistant accession tested. Expression of resistance in the intergeneric hybrid (L. esculentum cv. 'VF36' × S. lycopersicoides LA2951) suggested that resistance is at least partially dominant in tomato. A green fluorescent protein-tagged B. cinerea strain was used for confocal microscopic comparison of infection in leaves of S. lycopersicoides and tomato. Even though S. lycopersicoides supported spore germination, there was evidence for hyphal lysis and death 3 days after inoculation, at a time when lesions were expanding on susceptible tomato plants. The reduced frequency of B. cinerea lesion spread on S. lycopersicoides explains why this fungus produced fewer spores in this wild nightshade than in tomato.     

Mycoplasma bovis and otitis in dairy calves in the United Kingdom

Signs of severe otitis media in 20% of dairy calves on one farm were associated with Mycoplasma bovis infection, based on isolation from the external ear canal and nares. Affected calves seroconverted to M. bovis and no other significant bacteria were isolated. Infection was considered likely to have originated from cows in the milking herd based on evidence of seroconversion and detection of infection in a milk sample. M. bovis infection should be considered when investigating otitis problems in calves.     

Evaluation of mosquitoes, Aedes vexans, as biological vectors of porcine reproductive and respiratory syndrome virus

The objective of this study was to determine whether mosquitoes, Aedes vexans (Meigen), could serve as biological vectors of porcine reproductive and respiratory syndrome virus (PRRSV). Specifically, the study assessed the duration of viability and the site of PRRSV within mosquitoes, and evaluated whether PRRSV could be transmitted to a susceptible pig by mosquitoes following a 7- to 14-day incubation period after feeding on an infected pig. For the first experiment, a total of 100 mosquitoes were allowed to feed on a pig, experimentally infected with PRRSV (day 7 post-inoculation) and were then maintained alive under laboratory conditions. A set of 10 mosquitoes were collected at 0 hour (h), 6 h, 12 h, 24 h, 48 h, 72 h, 5 days (d), 7 d, 10 d, and 14 d post-feeding (pf). Samples of exterior surface washes, salivary glands, thorax carcasses, and gut homogenates were collected from each set of mosquitoes, and tested for PRRSV. Infectious PRRSV was detected by polymerase chain reaction and swine bioassay only from the gut homogenates of mosquitoes collected at 0 h and 6 h pf. For the second experiment, a total of 30 mosquitoes were allowed to feed on a pig, experimentally infected with PRRSV and the mosquitoes were then maintained under laboratory conditions. On each of day 7, 10, and 14 pf, a set of 10 mosquitoes were allowed to feed on a susceptible pig. Transmission of PRRSV to susceptible pigs did not occur, and PRRSV was not detected from the mosquitoes. These findings indicate that mosquitoes are not likely to serve as biological vectors of PRRSV.     

Resolution of a Left Ureteral Stone Using Electrohydraulic Lithotripsy in a Thoroughbred Colt

A 3-year-old Thoroughbred colt was presented for evaluation of azotemia and anorexia. Physical examination revealed a ureterolith in the left ureter, approximately 10 cm from the bladder, which was thought to obstruct urine flow by approximately 90% when viewed cystoscopically. Ultrasonographic examination of both kidneys revealed indistinct corticomedullary junctions, and the right kidney was more hyperechoic. A percutaneous biopsy of the right kidney revealed chronic interstitial nephritis with marked interstitial medullary fibrosis. Medical therapy consisting of IV fluids, sodium chloride PO, and ammonium chloride PO was initiated. Ureteroscopic electrohydraulic lithotripsy via a perineal urethrostomy was used to successfully remove the stone. Klebsiella oxytoca , which responded to oral enrofloxacin therapy, was cultured from the urine after surgery. Azotemia resolved and the horse resumed training.