Characterization of the ion channels formed by poliovirus in planar lipid membranes

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

Transrectal ultrasound applicator for prostate heating monitored using MRI thermometry

PURPOSE: For potential localized hyperthermia treatment of tumors within the prostate, an ultrasound applicator consisting entirely of nonmagnetic materials for use with magnetic resonance imaging (MRI) has been developed and tested on muscle tissue ex vivo and in vivo. METHODS AND MATERIALS: A partial-cylindrical intracavitary transducer consisting of 16 elements in a 4 x 4 pattern was constructed. It produced a radially propagating acoustic pressure field. Each element of this array (1.5 x 0.75 cm), operating at 1.5 MHz, could be separately powered to produce a desired energy deposition pattern within a target volume. Spatial and temporal temperature elevations were determined using the temperature-dependent proton resonant frequency (PRF) shift and phase subtraction of MR images acquired during ultrasonic heating. Four rabbits were exposed to the ultrasound to raise the local tissue temperature to 45 degrees C for 25 minutes. Six experiments compared thermocouple temperature results to PRF shift temperature results. RESULTS: The tests showed that the multi-element ultrasound applicator was MRI-compatible and allowed imaging during sonication. The induced temperature distribution could be controlled by monitoring the RF power to each transducer element. Therapeutic temperature elevations were easily achieved in vivo at power levels that were about 16% of the maximum system power. From the six thermocouple experiments, comparison between the thermocouple temperature and the PRF temperature yielded an average error of 0.34+/-0.36 degrees C. CONCLUSIONS: The MRI-compatible intracavitary applicator and driving system was able to control the ultrasound field and temperature pattern in vivo. MRI thermometry using the PRF shift can provide adequate temperature accuracy and stability for controlling the temperature distribution.     

The effect of benzodiazepines and flumazenil on motor cortical excitability in the human brain

In the present study, the effects of benzodiazepines (diazepam) were evaluated in terms of cortical excitability changes, as tested with transcranial magnetic simulation (TMS). In particular, analyzed were drug-induced changes regarding two selected parameters of TMS: (1) the cortical excitability threshold and (2) the silent period duration (SP). For this purpose, we evaluated the effects of long-term therapy with diazepam in the patients affected by anxiety disorders and the changes induced by single oral doses of diazepam in both healthy controls and patients. In addition, we tested cortical excitability changes in two 'extreme conditions' where a considerable concentration of serum benzodiazepine-like activity was reached, as represented by diazepam overdose and idiopathic recurrent stupor (IRS). In both groups of patients, a significant increment of motor threshold was found, while in the overdose patients, the SP was also increased. The administration of flumazenil in these two conditions was followed by a prompt reversal effect, consisting of a return to normal cortical excitability parameters. The long-term usage of diazepam in patients with anxiety disorders is associated with significantly increased threshold; the increased value of these parameters was temporarily further enhanced by the administration of a single oral dose of diazepam, which, in normal control subjects, is not associated with changes of cortical excitability. The results of this study reveal that different physio-pathological conditions induced by the influence of benzodiazepine and its antagonist are reflected in excitability changes which attest to the involvement and modification of cortical GABAergic activity.     

U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.     

Evaluation of a western blot system (DAVIH-BLOT) for the confirmation of HIV-1 antibodies]

Matrix metalloproteinases (MMPs), a sort of important enzymes involved in extracellular matrix metabolism, play critical roles in the process of tissues remodeling, wound healing and metastasis of tumors. Dot blot and in situ hybridization were used in this study to detect the expression and localization of MMP-9, an important proteolytic enzyme implicated in bone resorption in bone tissues. The results showed that the level of MMP-9 mRNA expression in osteoporotic bone tissues was significantly higher than that in normal control group and the cell types that expressed MMP-9 mRNA included mono- and multi-nuclear osteoclasts and some lining cells on the surface of bone matrix. It was suggested that MMP-9 play a key role in the development of bone loss in osteoporosis.     

Staphylococcal enterotoxin A-induced fever is associated with increased circulating levels of cytokines in rabbits

Rabbits were injected intravenously with 10 to 100 ng of staphylococcal enterotoxin A (SEA) per kg, and colonic temperatures were monitored. The febrile responses were compared with circulating levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-2, and IL-6 just before the injection of SEA. Both colonic temperatures and circulating levels of IFN, TNF, and IL-2 started to rise at 1 to 2 h and reached their peak levels at 3 to 5 h after SEA injection. Both the fever and the increased circulating levels of IFN, TNF, and IL-2 produced by SEA were decreased by pretreatment with indomethacin (a cyclo-oxygenase inhibitor) (15 mg/kg, intraperitoneally), anisomycin (a protein synthesis inhibitor) (15 mg/kg, subcutaneously), or dexamethasone (an effective anti-inflammatory and immunosuppressive agent) (4 mg/kg, intravenously) in rabbits. Rabbits were injected intravenously with 30 ng of SEA per kg on four consecutive days, and colonic temperatures were monitored. Compared to rabbits that received the single injection of SEA, rabbits that received four consecutive injections of SEA showed a lesser increase in circulating levels of IFN, TNF, and IL-2 as well as colonic temperatures in response to an intravenous dose of SEA (30 ng/kg). The data suggest that the prevention of the febrile response elicited by SEA by indomethacin, anisomycin, or dexamethasone is due to prevention by these compounds of the increase in the circulating levels of IFN, TNF, and IL-2. The pyrogenic hyporesponsiveness to repeated injection of SEA is associated with decreased production of these circulating cytokines.