An enhanced packaging system for helper-dependent herpes simplex virus vectors

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.     

Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans

Mutation in the Caenorhabditis elegans gene osm-6 was previously shown to result in defects in the ultrastructure of sensory cilia and defects in chemosensory and mechanosensory behaviors. We have cloned osm-6 by transposon tagging and transformation rescue and have identified molecular lesions associated with five osm-6 mutations. The osm-6 gene encodes a protein that is 40% identical in amino acid sequence to a predicted mammalian protein of unknown function. We fused osm-6 with the gene for green fluorescent protein (GFP); the fusion gene rescued the osm-6 mutant phenotype and showed accumulation of GFP in ciliated sensory neurons exclusively. The OSM-6::GFP protein was localized to cytoplasm, including processes and dendritic endings where sensory cilia are situated. Mutations in other genes known to cause ciliary defects led to changes in the appearance of OSM-6::GFP in dendritic endings or, in the case of daf-19, reduced OSM-6::GFP accumulation. We conclude from an analysis of genetic mosaics that osm-6 acts cell autonomously in affecting cilium structure.     

The nature of the spondylolytic defect. Demonstration of a communicating synovial pseudarthrosis in the pars interarticularis

The in vitro effects of angiotensin II (Ang II) in human vessels are not well studied. The development of specific Ang II-receptor antagonists has made it possible to delineate more carefully the receptor mechanisms involved. The objective of this study was twofold: to investigate the effect of Ang II on human coronary arteries and to study the effects of angiotensin II type 1 receptor blockade with losartan. The setting was contractile experiments with ring segments of coronary arteries. We observed that Ang II is a vasoconstrictor of human coronary arteries, with a pEC50 value of 9.26 +/- 0.22 and Emax of 68.7 +/- 9.61% of potassium-induced contraction. Losartan (10-100 nM) shifted the concentration-response curve of Ang II to the right, with pEC50 values of 7.64 +/- 0.10 and 7.00 +/- 0.15, respectively (p = 0.001), demonstrating the antagonistic properties of losartan. We also noted a decreased maximal response to Ang II after incubation of losartan, with Emax of 51.1 +/- 7.08% and 41.9 +/- 4.70% (p = 0.05), respectively. In conclusion, this is the first report describing the contractile effect of Ang II and the antagonizing effects of losartan in isolated human coronary arteries.     

Distribution of normal and abnormal fluid collections in the glenohumeral joint: implications for MR arthrography

OBJECTIVE: This study determined levels of cathepsin D activity in tissue components of normal human ovary to establish a basis for comparison with human ovarian adenocarcinomas. METHODS: Cathepsin D activity per mg tissue, per microgram protein, and per microgram DNA was determined in human ovarian tissues (cortex, follicle, corpus luteum, corpus albicans) from patients of various ages and during the menstrual cycle. Levels of cathepsin D activity were also determined in ovarian adenocarcinomas and other pathologic tissues. RESULTS: Cathepsin D levels (per mg tissue) were significantly greater (P < .001) in ovarian follicle and corpus luteum compared with cortex. Although there was not a clear correlation between enzyme activity in the cortex and day of the menstrual cycle or patient age, levels of enzyme activity appeared to decrease with each parameter. Cathepsin D levels per mg tissue in ovarian adenocarcinoma were 40% higher than in postmenopausal ovarian cortex, but the difference was not statistically significant. CONCLUSION: The diversity of cathepsin D levels in normal ovarian tissue compartments indicates that specific tissues must be used in comparisons with ovarian tumors.     

Study of hemostasis in pediatric patients with portal vein thrombosis

We describe the behavior of hemostatic variables in children with portal vein thrombosis (PVT) and in a control pediatric population. Hereditary protein C (PC) or protein S (PS) deficiency was not a etiologic factor for PVT in children. Minor signs of consumption of coagulation factors II, V, fibrinogen and hyperfibrinolysis were detected. One child had lupus anticoagulant (LA).     

Effects of luminance, contrast, and blur on visual acuity

Although previous investigations have reported that changes in background luminance, stimulus contrast, and dioptric blur can each affect visual acuity independently, it has not been shown how these three variables interact to influence visual acuity. This is a particularly important issue if one is interested in predicting how individuals with different refractive characteristics will be able to perform acuity-based tasks in degraded visual environments with low background lighting and contrast levels. To investigate these relations, we conducted a series of experiments in which measurements of visual acuity were obtained for four subjects using Landolt C targets of varying contrast at several background luminances for levels of blur between 0 and 8 diopters (D). Our results show that visual acuity is significantly affected by all three factors, and that their effects are essentially additive. At all luminance and contrast levels, the reduction in visual acuity is greatest for dioptric blur up to 2.0 D, with a more gradual reduction in visual acuity for dioptric blur of greater than 2.0 D. At all blur and luminance levels, visual acuity decreases gradually for contrast levels down to 20%, and decreases sharply for lower contrast levels. Over the range of background luminances we tested (75.0 to 0.075 cd/m2), visual acuity decreases linearly with reductions in luminance. The additive effects of dioptric blur, contrast, and luminance provide a basis for predicting visual acuity-related task performance for individuals in different visual environments. For example, an individual with 6/6 (20/20) visual acuity under high luminance-high contrast conditions will fall to 6/18 (20/60) acuity for low luminance conditions and 6/30 (20/100) for low luminance-low contrast conditions. Similarly, an individual with an uncorrected visual acuity of 6/30 (20/100) under optimal conditions will fall to approximately 6/120 (20/400) under low luminance conditions and 6/240 (20/800) under low luminance-low contrast conditions.     

Recording eye movements using coaxial cameras--applications for visual ergonomics and reading studies

We developed a system of coaxial video cameras that records monocular eye position and scene, and superimposes these images using a digital video mixer. We mounted miniature video cameras above and below a cube beam-splitting prism in the spectacle plane. An infrared emitting diode was imaged in the cornea to locate eye position. The technique was accurate to about 0.5 degrees within 15 degrees of primary gaze; however, we see its main advantages as being its low cost and simple design that, for some applications, does not require complex computer analysis and data manipulation. With improved camera optics, it has the potential for helmet mounting and use remote from a recording console. We used the instrument to monitor a reader's eye position when using low vision devices, and see applications of the technique in the field of visual ergonomics and sports vision.     

Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa

It is widely agreed that after two or more seizures patients should be given antiepileptic treatment, but there is still controversy about the treatment of patients after a first unprovoked seizure. In a multicenter, randomized, open trial, patients with a first tonic-clonic seizure were randomized to immediate treatment (carbamazepine, phenytoin, phenobarbital, or sodium valproate) or to treatment only after another seizure. Fifty-two (24%) of the 215 patients randomized to immediate treatment and 85 (42%) of the 204 randomized to delayed treatment experienced seizure recurrence during follow-up. Age, acute treatment of the seizure with benzodiazepines, remote etiologic factors, and EEG abnormalities were significant predictors of relapse. Of the immediately treated patients, 87% had no seizures for a year and 68% had no seizures for 2 years, whereas only slightly fewer initially untreated patients (83% and 60%) achieved these endpoints. Patients treated after the first seizure and those treated after seizure relapse had the same time-dependent probability of achieving 1 and 2 seizure-free years. None of the variables that were prognostic predictors of relapse was significantly associated with the probability of having 1 or 2 years of seizure control. Anticonvulsants in patients presenting a first tonic-clonic seizure reduce the risk of relapse; however, 50% of patients who are not treated will never experience a second seizure. Moreover, the probability of long-term remission is not influenced by treatment of the first seizure.