DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) respiratory infection. A single intramuscular injection with plasmid DNA encoding EHV-1 glycoprotein D (EHV-1 gD), including its predicted C-terminal membrane anchor sequence, induced a specific antibody response detectable by 2 weeks and maintained through 23 weeks post injection. A second injection at 4 weeks markedly enhanced the antibody response and all EHV-1 gD-injected mice developed neutralizing antibodies. A lymphocyte proliferative response to whole EHV-1 was observed and a predominance of IgG2a antibodies after DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunized with EHV-1 gD DNA were able to clear virus significantly more rapidly from lung tissue and showed reduced lung pathology, in comparison to control mice.     

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae

A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K(+)-selective current whose attributes resemble the leak conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.     

Microemboli during carotid angiography. Association with stroke risk factors or subsequent magnetic resonance imaging changes?

BACKGROUND AND PURPOSE: Carotid angiography is associated with a 1% risk of major stroke. Recently, transcranial Doppler ultrasonography (TCD) has shown cerebral microemboli during carotid angiography. To determine their significance, we correlated the number of microemboli during angiography with clinical characteristics, angiography findings, and preangiography and postangiography cerebral magnetic resonance imaging (MRI). METHODS: One middle cerebral artery was monitored with TCD in 24 patients during angiography for carotid territory ischemia. The number of microemboli was correlated with angiographic and clinical characteristics. T2-weighted cerebral MRI was performed before and < or = 48 hours after angiography, and the number of new ischemic lesions was determined in a blinded review. RESULTS: Microemboli were seen in all patients, with an average of 51 per procedure (range, 12 to 154). The majority of microemboli had signal characteristics typical of air. Sixteen of the 24 patients had both preangiography and postangiography MRI. One of 24 patients had an angiographic stroke, with a single new thalamic lesion on MRI. No other patient had a new lesion. The microembolus count correlated with the angiographic contrast volume (P < .001) but not with any other radiological or clinical characteristic. CONCLUSIONS: This study confirmed the presence of numerous cerebral microemboli during carotid angiography. The microembolic signal characteristics and the correlation with contrast volume indicate that introduced air is the cause. These microemboli are usually clinically silent and do not lead to new changes on cerebral MRI.     

Band 3 protein: structure, flexibility and function

The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10(-8) and 4 x 10(-7) M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10(-5) and 7 x 10(-3) M. The within-run precision was +/- 5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.     

Exon scanning for mutation of the NF2 gene in schwannomas

Family studies and tumor analyses have combined to indicate that neurofibromatosis 2 (NF2), a disorder characterized by multiple benign tumors of the nervous system, and sporadic non-inherited forms of the same tumor types are both caused by inactivation of a tumor suppressor gene located in 22q12. Recently, the gene encoding merlin, a novel member of a family of cytoskeleton-associated proteins, was identified as the NF2 tumor suppressor. To facilitate the search for merlin mutations, we have defined the exon-intron boundaries for all 17 NF2 exons, including one subject to alternative splicing. We have developed polymerase chain reaction assays to amplify each exon from genomic DNA, and used these assays to perform single-strand conformation polymorphism analysis of DNA from 30 sporadic and eight NF2-derived schwannomas, the hallmark tumor type in this disorder. Of a maximum of 60 alleles scanned, 32 showed mutations affecting expression of the merlin protein. Thirty of these mutations are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal splicing, while two are missense mutations. Thus, inactivation of merlin is a common feature underlying both inherited and sporadic forms of schwannoma.     

Magnetic resonance coronary angiography in heart transplant recipients

BACKGROUND: Magnetic resonance angiography (MRA) using segmented k-space fast low-angle shot imaging has recently been used to demonstrate the proximal coronary arteries in healthy subjects and in patients with coronary artery disease. We assessed the sensitivity and specificity of coronary MRA in heart transplant recipients and investigated the feasibility of coronary MRA in patients with metallic sutures and clips in the chest. MATERIALS AND METHODS: Sixteen cardiac transplant patients aged 57.2 +/- 7.9 years (mean +/- SD) were recruited. Forty-eight arterial segments were evaluated, including the left main artery (LMA), left anterior descending artery (LADA) and right coronary artery (RCA). We excluded the left circumflex artery which could not be imaged accurately. The average time between heart transplant operation and MRA was 6 years, whereas that between MRA and X-ray angiography was 4 months. The coronary MRA was interpreted by two experienced investigators who were blinded to the coronary X-ray angiography results. Similarly, the coronary X-ray angiography results were interpreted by two experienced investigators blinded to the MRA results. The coronary arterial segments were classified by MRA as being normal or as having an amount of disease that was significant (> 50% lesion) or insignificant (< 50% lesion). RESULTS: There were 28 true-negative, five true-positive, four false-negative and six false-positive results. Of the 28 true-negative cases, 13 were in the LMA, six in the LADA and nine in the RCA. There was one false-positive LMA, two false-positive LADA and three false-positive RCA stenoses. There were four false-negative results in the LADA and one in the RCA. Clips precluded evaluation in one LMA, one LADA and one RCA. One LMA and one LADA were not evaluated as a result of poor images. One false-positive RCA stenosis was caused by a metallic clip. Three of the false-negative LADA stenoses had lesions in the distal third of the artery. The sensitivity, specificity, negative and positive predictive values were generally poor for the left coronary artery. The best results were for the RCA (sensitivity 100%, specificity 75%, positive predictive value 50% and negative predictive value 100%). The specificity in the left coronary arteries (LMA and LADA) was 86%, but the other indicators were all poorer. For the RCA, LMA and LADA combined, the overall sensitivity was 56%, specificity 82%, predictive accuracy 45% and negative predictive value 88%. In three patients, < 50% RCA lesions were seen in the MRA data, which were all confirmed by angiography. No < 50% lesions were seen in the LMA or in the LADA by MRA or by X-ray angiography. CONCLUSION: Coronary MRA using the segmented fast low-angle shot technique is feasible in heart transplant recipients but the sensitivity and specificity of this method are limited. Further developments in coil design, rapid imaging techniques and respiratory monitoring methods are necessary to improve the accuracy of coronary MRA.     

Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma

About 30% of human plasma protein C (PC) is of lower molecular weight than the predominant alpha-form. The minor beta-form arises as a consequence of the lack of glycosylation at Asn329. Although the functional role of Asn329 has been investigated by in vitro mutagenesis, until now no naturally occurring mutations have been reported at this site. We describe here the case of two identical twin sisters compound heterozygous for two novel PC mutations: Cys78-->Stop inherited from the maternal side and Asn329-->Thr inherited from the paternal side, associated with the presence of only the beta-form of PC in plasma. The Cys78-->Stop substitution is predicted to abolish PC synthesis from one allele, whereas the Asn329-->Thr substitution results in the reduced synthesis of a beta PC variant with decreased functional activity. PCN329T from the two monovular twin sisters was purified and its active form APCN329T was assessed for its ability to inactivate factor Va. Whereas no differences were observed between the activation rates of normal PC and PCN329T, APCN329T inactivated human factor Va with a rate slower than the normal APC. This is the first report of a PC defect involving glycosylation of the molecule. This defect results in the presence of only the beta-form of PC in human plasma and is responsible for the reduced anticoagulant activity observed.     

Regional audit of pedestrian accident care

In all, 160 serious pedestrian accidents (ISS > 15 or death), were recorded during a 12-month prospective study of all trauma in a population of 3.2 million. Of these, 35 died at scene, 125 arrived at hospital alive and 68 (54 per cent) subsequently died. There were 35 (22 per cent) children, and 62 per cent (39) were more than 60 years of age. Prehospital care significantly delayed transfer to hospital. In the accident and emergency department, only 38 per cent of those unconscious had a cervical collar applied, and only 67 per cent were intubated. Of those transferred for neurosurgical care, 34 per cent were not intubated. The Median Injury Severity Score for each outcome group was similar between age groups. The Revised Trauma Score and APACHE II score showed significant differences between those who lived and died. TRISS analysis revealed that 32 per cent of deaths and 12 per cent of survivors were unexpected. ATLS treatment protocols should be instituted for prehospital care and in all accident and emergency departments (A&E).