Phi29 pRNA vector for efficient escort of hammerhead ribozyme targeting survivin in multiple cancer cells

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme escorted by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5'/3' helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganizes into a circularly permuted form, and the 5'/3' ends are relocated and buried in the original 71'/75' positions. Effective silencing of the anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozyme's strength in medical application.     

Surgical treatment of medically refractory epilepsy in childhood

Twenty-five percent of children with epilepsy continue to seize despite the best medical management and may be defined as medically refractory. Many children with medically refractory localization-related epilepsy, i.e. seizures which originate in a particular area of the brain and secondarily spread to involve other brain regions, may benefit from a variety of surgical treatments including hemispherectomy, corpus callosotomy, focal cortical resection of the temporal lobe, focal cortical resection of extratemporal regions of the brain, and multiple subpial resections. A successful outcome from epilepsy surgery is generally defined as a seizure-free state with no imposition of neurologic deficit. In order to achieve these twin goals two criteria must be fulfilled. First, precise localization of the epileptogenic zone in the brain is necessary. The epileptogenic zone may be defined as the region of epileptogenic cerebral cortex whose removal will result in a seizure-free state. Second, one must determine the anatomic localization of eloquent cortex in the brain in order to spare these areas during any planned cortical excision of epileptogenic cortex. Several diagnostic measures may be used to achieve a successful surgical outcome. A clinical history to ascertain the earliest symptom in the clinical progression of the seizure (semiology) is imperative as is ictal and interictal scalp EEG, neuropsychological testing, magnetic resonance imaging, positron emission tomography, single photon emission computerized tomography, and interictal magnetoencephalography. In the typical child undergoing evaluation for epilepsy surgery, if the clinical, neuropsychological, EEG, and radiological data are all concordant and point to the same area of epileptogenicity in the brain, cortical excision of the suspected epileptogenic zone is undertaken. However, if the data are discordant, and/or the epileptogenic zone resides wholly or in part within eloquent cortex, invasive intracranial monitoring from depth and/or subdural electrodes during a seizure is required to map out the areas of epileptogenicity in the brain. The assessment of potential risks and benefits for this type of epilepsy surgery in children involves complex age-related issues, including the possible impact of uncontrolled seizures, medication, or surgery on learning and development.     

Natural history of multiple hereditary osteochondromatosis of the lower extremity and ankle.

In this study the authors evaluated the natural history of the ankle joint in patients with multiple hereditary osteochondromatosis. Thirty-eight subjects with an average age of 42 years completed a detailed subjective questionnaire and underwent clinical and radiographic evaluation of their ankles. Three subjects (8%) indicated their ankle involvement affected their vocation, and 12 (32%) were limited in recreational sports. Seven patients (18%) had pain in at least one ankle on a weekly basis, with an average ankle pain score of 2.2. Ankle range of motion averaged 50 degrees and subtalar motion was considered normal in two thirds of ankles. Radiographic evaluation documented an average tibiotalar tilt of 9 degrees of ankle valgus, with evidence of degenerative joint disease noted in 14 ankles (19%). Those with arthritic changes had significantly more tibiotalar tilt and diminished ankle range of motion compared with those without radiographic signs of osteoarthritis. These findings document measurable decreases in ankle function and suggest that correction or prevention of excessive tibiotalar tilt may be warranted to improve outcome.     

Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated intravascular coagulation

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A- peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.     

Continuous potential display of ictal electrocorticography.

The objective of this study was to determine whether the animation of electrical activity recorded on ictal electrocorticograms (ECoGs) can demonstrate the propagation of seizure discharges from the epileptogenic zone (EZ) to the surrounding cortical area. A computer program, continuous potential display (CPD), was designed to animate the color-coded potential changes in 5-msec intervals at each recorded site. This program was used to analyze 35 ictal ECoGs recorded by subdural grid electrodes from 11 subjects who underwent epilepsy surgery for intractable partial seizures. Continuous potential display demonstrated recurrent cycles of seizure propagation from the EZ to the surrounding cortical area even when seizure discharges appeared widespread on ECoG. Hence, the EZ could be mapped at any time during the seizure course. The EZ mapped by analyzing a small fraction of ECoG during widespread seizure discharges using CPD only overlapped 69 +/- 24% (mean +/- standard deviation) of the surgical area. The EZ mapped by CPD had 34 +/- 22% false positives and 35 +/- 27% false negatives. Animation of potential changes recorded by ictal ECoG can assist in studying the temporal and spatial patterns of seizure propagation and in mapping the EZ for surgical resection.     

Epidermal growth factor transcription, translation, and signal transduction by rat type II pneumocytes in culture.

Epidermal growth factor (EGF) is known to induce fetal lung maturation and its receptor is present in the lungs of several species. Recently, EGF has been immunolocalized in type II pneumocytes in rat lung. We postulated that EGF is synthesized in type II pneumocytes and that, because of its position-restricted distribution within the alveolus, EGF might act as an autocrine regulator of type II pneumocyte function. Herein, we have tested the hypothesis using adult rat type II pneumocytes in primary culture. In situ hybridization, using an oligonucleotide probe corresponding to amino acid residues 1070 to 1081 of mouse EGF precursor, demonstrated the presence of EGF precursor mRNA. Upon S-200 Sephacryl gel chromatography of type II pneumocyte extracts, EGF-reactive protein eluted as a high-molecular-weight form (greater than 100 kD). EGF immunoreactivity was localized within type II pneumocytes in the periphery of groups of 10 to 15 cells in culture. The type II pneumocytes bound [125I]EGF in a specific manner, indicating the presence of EGF receptors. Scatchard plots gave an apparent affinity constant (Ka) of 1 x 10(9) liters/mol, and the number of receptors was estimated to be 4.8 x 10(11) mg protein (50 per cell). EGF receptor binding specificity was confirmed by the absence of an autoradiographic signal for cells incubated in the presence of a 100-fold excess concentration of transforming growth factor-alpha. Binding of [125I]EGF could also be downregulated 95% by incubation with 0.2 nM transforming growth factor-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-Hydroxybutyric acid binding sites: evidence for coupling to a chloride anion channel

The effect of eight anions, including chloride, on the binding of gamma-hydroxy[2,3-3H]butyric acid (GHB) to synaptosomal membranes of rat and human brain was ascertained, as was the effect of a number of other allosteric modulators of the GABA/benzodiazepine/picrotoxin complex. All ions which were active at the chloride ion channel, inhibited the binding of [3H]GHB in a dose-dependent manner, with maximum inhibition of binding being 60% of 300 mM concentration of anion. Inactive ions in this binding system included sulfate, acetate and fluoride, all impermeable to the chloride ion channel. The inhibition of binding was temperature-dependent, being abolished at 37 degrees C and was independent of the cation used. The binding of [3H]GHB was also enhanced by pentobarbital, picrotoxin and diazepam but unchanged in the presence of GABA, muscimol, bicuculline, baclofen or strychnine. These data raise the possibility that the epileptogenic effect of GHB may be modulated by an action on the chloride ion channel, that is tightly coupled to the GABA/benzodiazepine/picrotoxin and/or GHB receptor complex.     

Negative predictive value of a vision screening program aimed at children aged 3 to 4 years old

PURPOSE: Nova Scotia has a vision screening program which assesses children aged 4[1\2] to 5[1\2] years. However, its use in younger children proved impossible. This study will examine a modified screening protocol for the younger children (3 to 4 years old) and determine its negative predictive value and minimum age for reliable application. MATERIALS AND METHODS: Public health nurses administered the study protocol to 3- to 4-year-old children. One hundred seventy-eight children were screened over two summers. Medical and family history, external inspection, as well as measures of visual acuity with the Lea Hyvarinen symbols chart and stereoacuity with Frisby plates were recorded. Results were compared with a gold standard examination that included full orthoptic and ophthalmologic evaluations. One hundred forty-one (79%) children underwent the gold standard examination. Agreement between screening and gold standard examinations was studied. RESULTS: Data showed increased concordance between screening and gold standard examination results with increasing age up to 41 months. Negative predictive value (NPV) and specificity also improved when data were separated by this age. In children <41 months old, the screening test NPV was 90%, specificity, 68%, and sensitivity, 75%. In comparison, children >/=41 months old had screening test NPV of 96%, specificity, 95%, and sensitivity, 50%. Specificity was higher in the older age group ( P < 0.001). Sensitivity was lower ( P = 0.004). CONCLUSION: This study's vision screening protocol appears better suited for children 41 months and older. They had better pass/fail reproducibility than children <41 months. The test's simplicity allows easy use by non-eye-care professionals. It could potentially lower the reliable screening age of children by 13 months, from 54 months of age (4[1\2] years old) to 41 months. This screening may miss some refractive errors and microtropia/monofixation syndrome, despite normal visual acuity, stereoacuity, and external inspection.