Association between time to treatment and functional outcomes according to the Diffusion-Weighted Imaging Alberta Stroke Program Early Computed Tomography Score in endovascular stroke therapy.

BACKGROUND AND PURPOSE: The rate at which the chance of a good outcome of endovascular stroke therapy (EVT) decays with time when eligible patients are selected by baseline diffusion-weighted magnetic resonance imaging (DWI-MRI) and whether ischaemic core size affects this rate remain to be investigated. METHODS: This study analyses a prospective multicentre registry of stroke patients treated with EVT based on pretreatment DWI-MRI that was categorized into three groups: small [Diffusion-Weighted Imaging Alberta Stroke Program Early Computed Tomography Score (DWI-ASPECTS)] (8-10), moderate (5-7) and large (<5) cores. The main outcome was a good outcome at 90 days (modified Rankin Scale 0-2). The interaction between onset-to-groin puncture time (OTP) and DWI-ASPECTS categories regarding functional outcomes was investigated. RESULTS: Ultimately, 985 patients (age 69 ± 11 years; male 55%) were analysed. Potential interaction effects between the DWI-ASPECTS categories and OTP on a good outcome at 90 days were observed (Pinteraction  = 0.06). Every 60-min delay in OTP was associated with a 16% reduced likelihood of a good outcome at 90 days amongst patients with large cores, although no associations were observed amongst patients with small to moderate cores. Interestingly, the adjusted rates of a good outcome at 90 days steeply declined between 65 and 213 min of OTP and then remained smooth throughout 24 h of OTP (Pnonlinearity  = 0.15). CONCLUSIONS: Our study showed that the probability of a good outcome after EVT nonlinearly decreased, with a steeper decline at earlier OTP than at later OTP. Discrepant effects of OTP on functional outcomes by baseline DWI-ASPECTS categories were observed. Thus, different strategies for EVT based on time and ischaemic core size are warranted.

Tracking the startle response of guppies Poecilia reticulata in three dimensions.

A three-dimensional analysis of startle behaviours of guppies Poecilia reticulata, in dyads or alone, from two populations that show distinct differences in shoaling behaviour was performed. During the first few seconds after a startling stimulus, changes in behaviour, which could be critical if an individual is to survive a predatory attack, and the interactions between pairs of P. reticulata were examined. The enhanced social interactions immediately after the stimulus, as a proxy for shoaling behaviour, and their dissipation were quantified. Social (individuals tested in dyads) v. asocial (tested alone) responses to the startling stimulus were also compared. The three-dimensional reconstruction, from a two-camera, high-frame-rate tracking system allowed for the tracking of the individuals' speed and speed recovery and, for P. reticulata in dyads, interindividual distance and orientation. For the dyads from the high-predation population, the closer the individuals were to each other, the more likely they were to be parallel, but no correlation was found for the low-predation P. reticulata. The startle response of P. reticulata comprised the following sequence: freezing, darting and skittering and recovery to pre-stimulus swimming behaviour. Upon repeated encounters with the stimulus, a reduced shoaling and startle response was observed, although the rate of reduction was faster in P. reticulata from the high-predation population than those from the low-predation population. The results are discussed in light of what is known about the anti-predator behaviour of this species.

Association between metabolic syndrome and functional outcome in patients with acute ischaemic stroke.

BACKGROUND AND PURPOSE: There is a paucity of information on the role of metabolic syndrome (MetS) as a prognosticator after ischaemic stroke. We investigated the association between MetS and functional outcome in patients with acute ischaemic stroke. METHODS: We evaluated 691 consecutive patients with acute stroke who were admitted to a tertiary medical center between January 2007 and June 2011. We defined MetS as having three or more of the five cardinal cardiovascular risk factors. Unfavorable functional outcome was determined using responder analysis, in which the outcome was adjusted by the initial severity of the stroke. Multivariable logistic regression analysis was used to evaluate the relationship between MetS and unfavorable outcomes (UnFO). RESULTS: Among 691 patients, 277 patients were classified as having an UnFO. The association between MetS and UnFO remained significant after adjusting for possible confounders; the adjusted odds ratio (95% confidence interval) was 1.57 (1.13-2.19). The risk for UnFO was positively associated with the number of MetS components. CONCLUSIONS: MetS may be a potent predictor of functional outcome after ischaemic stroke.

Topical administration of the pan-Src kinase inhibitors, dasatinib and LCB 03-0110, prevents allergic contact dermatitis in mice.

BACKGROUND: Allergic contact dermatitis (ACD) is a delayed type of T cell-mediated cutaneous inflammatory response, in which multiple cell types are involved. Dasatinib and LCB 03-0110 are small molecule multityrosine kinase inhibitors, and they share remarkably similar target kinases such as the c-Src family, Btk and Syk, which play key roles in the cell signalling of T cells and other inflammatory cells. OBJECTIVES: To test the anti-ACD activity of dasatinib and LCB 03-0110 and compare it with that of tacrolimus (FK506) and triamcinolone acetonide (a glucocorticoid), which are widely used for topical treatment of ACD, and to examine the two compounds for their capacity to induce skin atrophy, a side-effect. METHODS: ACD was induced on the ears of mice by repeated topical application of oxazolone. Each test compound was then topically applied on the ear. Ear swelling, epidermal thickness and levels of inflammatory cytokines were measured. The skin atrophy induced by the compounds was tested during prolonged application on the dorsal skin of hairless mice, followed by haematoxylin and eosin staining. RESULTS: Dasatinib and LCB 03-0110 suppressed the symptoms of ACD such as ear swelling, increase in epidermal thickness and synthesis of inflammatory cytokines (i.e. interleukin-1ß, tumour necrosis factor-α and interferon-γ) in a dose-dependent manner. The two compounds showed near-equal potency to tacrolimus; however, their potency was lower than that of triamcinolone acetonide. Prolonged treatment with the two compounds did not induce any skin atrophy, whereas use of steroidal agents induced severe atrophy. CONCLUSIONS: Dasatinib and LCB 03-0110 could be used as effective agents for the treatment of ACD without the adverse side-effect of skin atrophy.

Effects of celecoxib on hematoma and edema volumes in primary intracerebral hemorrhage: a multicenter randomized controlled trial.

BACKGROUND AND PURPOSE: We investigated the effect of celecoxib, a selective inhibitor of cyclo-oxygenase 2, in patients with intracerebral hemorrhage (ICH). METHODS: We conducted a multicenter, randomized, controlled, and open with blinded end-point trial of 44 Korean patients 18 years or older with ICH within 24 h of onset. The intervention group (n = 20) received celecoxib (400 mg twice a day) for 14 days. The control group (n = 24) received the standard medical treatment for ICH. The primary end-point was the number of patients with a change in the volume of perihematomal edema (PHE) from the 1st to the 7th ± 1 day (cut-off value, 20%). RESULTS: The time from onset to computed tomography scan slightly differed between groups (177 ± 160 min for control vs. 297 ± 305 min for the celecoxib group; P = 0.10). In the primary end-point analysis using cut-off values, there was a significant shift to reduced expansion of PHE in the celecoxib group (P = 0.005). With respect to the secondary end-points, there was also a significant shift to reduced expansion of ICH in the celecoxib group (P = 0.046). In addition, the expansion rate of PHE at follow-up tended to be higher in the control group than in the celecoxib group (90.6 ± 91.7% vs. 44.4 ± 64.9%; P = 0.058). CONCLUSIONS: In our small, pilot trial, administration of celecoxib in the acute stage of ICH was associated with a smaller expansion of PHE than that observed in controls.

Hepatitis B virus X protein enhances Myc stability by inhibiting SCF(Skp2) ubiquitin E3 ligase-mediated Myc ubiquitination and contributes to oncogenesis.

The stability of Myc oncoprotein is regulated by Skp/cullin/F-box (SCF) ubiquitin E3 ligase, in particular, via either SCF(Skp2) or SCF(Fbw7) ubiquitin E3 ligases. An earlier study has shown that hepatitis B virus X protein (HBx) augments Myc stability via a mechanism involving the inhibition of Myc ubiquitination. However, the underlying mechanism by which HBx inhibits Myc ubiquitination remained to be elucidated. Moreover, to what extent HBx-mediated Myc stabilization contributes to viral oncogenesis is unknown. First, we corroborated the physiological significance of HBx-mediated Myc stabilization in HBV-replicating cells by demonstrating that (1) the elevation of Myc level in a HBV-replicating HepG2.2.15 cell compared to parental cells; (2) HBx-mediated Myc stabilization in a HBV replicon transfected cells; and (3) the inhibition of Myc ubiquitination by HBx in a HBV replicon transfected cells. Then, the molecular interaction between HBx and Myc protein was revealed via coimmunoprecipitation and immunofluorescence experiments. Subsequent analysis indicated that HBx stabilizes Myc oncoprotein by blocking Skp2, as opposed to Fbw7, -mediated Myc ubiquitination. Next, we defined the Myc-binding region to four residues (VFVL) near the C-terminus of HBx polypeptide. An HBx variant with mutated VFVL consistently failed to not only interact with Myc but also suppress Myc ubiquitination. Importantly, the VFVL mutant lost the ability to transform NIH3T3 cells in concert with Ras, implying that the HBx-Myc interaction is critical for viral oncogenesis. Consistently, immunohistochemistry of liver biopsies revealed that Myc protein is elevated in HBV-infected tissues. We concluded that HBx stabilizes Myc oncoprotein by inhibiting SCF(Skp2) ubiquitin E3 ligase-mediated Myc ubiquitination, and the HBx-mediated Myc stabilization greatly contributes to viral oncogenesis.

Activated ras oncogene collaborates with HBx gene of hepatitis B virus to transform cells by suppressing HBx-mediated apoptosis.

The hepatitis B virus HBx protein is a promiscuous transactivator implicated in the development of hepatocellular carcinoma. The ectopic expression of HBx fails to transform both primary and immortalized rodent cells, but rather induces apoptosis. Furthermore, most transgenic mice harboring HBx do not develop liver tumors. Thus, it remains unclear whether and how HBx contributes to oncogenesis. Here, we show that HBx collaborates with activated H-ras to transform immortalized rodent cells. Indeed, REF52 cells transfected by both HBx and activated H-ras were morphologically transformed and were able to grow in soft agar. Remarkably, nude mice injected with REF52 cells transfected by both HBx and activated H-ras developed tumors, whereas the mice injected with REF52 cells transfected by either gene alone did not. Thus, we concluded that HBx could contribute to neoplastic transformation of cells in collaboration with other oncogenes, such as H-ras, that renders cells to overcome the HBx-mediated apoptosis. Further, we found that HBx mediated apoptosis was suppressed by activated H-ras through activation of the phosphatidylinositol-3 kinase and Akt pathway. Data presented here firmly established the oncogenic potential of HBx during multistage carcinogenesis. Oncogene (2001) 20, 16 - 23.

Expression of cyclooxygenase-2 (COX-2) in hepatocellular carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor, NS-398.

Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. In hepatocellular carcinoma (HCC), the expression pattern of COX-2 protein has been well correlated with the differentiation grade, suggesting that abnormal COX-2 expression plays an important role in hepatocarcinogenesis. We investigated the expression pattern and clinical significance of COX-2 in HCC tissues. In addition, we evaluated the efficacy of a selective COX-2 inhibitor, NS-398, in three hepatoma cell lines. Thirty-six HCC tissues, 15 hepatoma cell lines, 1 colorectal cell line (HT-29), and 1 fibroblast cell line (SV80) were included in the study. We evaluated serological tests and histological and radiological evaluations of HCC tissues. Immunohistochemical staining for COX-2 was performed on 36 HCC tissues and 17 cancer cell lines. A cell viability assay for growth inhibition of NS-398 in five cell lines was performed. Immunohistochemically, all six well-differentiated HCCs were positive, whereas 83% (10 of 12) of the poorly differentiated HCCs were negative. There was no significant relationship between the intensity of COX-2 expression and the level of alpha-fetoprotein, tumor size, presence of portal vein thrombosis, tumor capsule and metastasis, Tumor-Node-Metastasis staging, and growth types (P > 0.05). According to the cell viability assay, NS-398 suppressed the growth of all cell lines, independent of the degree of COX-2 expression. The inhibitory effect on each cell line was identified in 10 microM NS-398 and was significantly strong in 100 microM NS-398. All cell lines exhibited apoptosis, which was identified by 4'-6-diamidino-2-phenylindole staining. In conclusion, COX-2 may be a determinant of the differentiation grade of HCC, and the inhibition of COX-2 can induce growth suppression of hepatoma cell lines via induction of apoptosis.

Effect of hormone replacement therapy on lipoprotein(a) and lipid levels in postmenopausal women. Influence of various progestogens and duration of therapy.

BACKGROUND: Estrogen replacement therapy in postmenopausal women reduces the risk of coronary artery disease. One of the possible mechanisms of this effect is the modification of lipid profiles. However, there is controversy concerning the effects on lipoprotein(a) [Lp (a)] and lipid levels of progestogens administered with estrogen. METHODS: Five hundred fifty-one postmenopausal women were divided into 5 groups: group 1, 0.625 mg of conjugated equine estrogen (CEE) (n = 140); group 2, 0.625 mg of CEE plus 5 mg of medroxyprogesterone acetate (MPA) (n = 97); group 3, 0.625 mg of CEE plus 10 mg of MPA (n = 109); group 4, 2 mg of estradiol valerate plus 0.5 mg of norgestrel (n = 134); and group 5, control (n = 71). The Lp(a) and lipid levels were measured before and 2, 6, and 12 months after hormone replacement therapy. RESULTS: Estrogen replacement therapy for 12 months lowered the Lp(a) level by 37.1%. The addition of progestogen attenuated the Lp(a)-lowering effect of estrogen. The high-density lipoprotein cholesterol (HDL-C) level was markedly increased in group 1 (16.5%), was moderately increased in groups 2 (10.8%) and 3 (11.3%), and was not changed in group 4. The low-density lipoprotein cholesterol level was decreased by 10.9% to 17.6% in all the treatment groups. Estrogen replacement therapy for 2, 6, and 12 months raised the HDL-C level by 7.2%, 17.4%, and 17.8%, respectively. In the group with combined estradiol plus norgestrel therapy, the HDL-C level was decreased after 2 months and was not changed after 6 and 12 months. The groups that received CEE plus MPA showed intermediate effects between the group that received CEE only and the group that received estradiol plus norgestrel. CONCLUSIONS: Combined estrogen and progestogen therapy may have effects on the heart different from those of estrogen therapy alone because of adverse impact of progestogens on Lp(a) and HDL-C levels. The effects of progesterones were dependent on the androgenic potency of progestogen and the duration of therapy.

Changes in Lp(a) lipoprotein and lipid levels after cessation of female sex hormone production and estrogen replacement therapy.

OBJECTIVE: To investigate the serial changes in Lp(a) lipoprotein levels with the loss of female sex hormones by surgical menopause and with estrogen replacement therapy in the same woman. PATIENTS AND METHODS: Forty-four premenopausal women who underwent a transabdominal hysterectomy (TAH) because of benign gynecological disorders were divided into two groups: women who underwent a TAH and unilateral salpingo-oophorectomy (n=31) and women who underwent a TAH and bilateral salpingo-oophorectomy (n=13). In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, 0.625 mg of conjugated equine estrogen was given daily 2 months after the operation. The levels of Lp(a) lipoprotein and lipids were measured before and at 2 and 4 months after the operation. RESULTS: In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the mean (+/-SD) concentration of Lp(a) lipoprotein was increased by 24.5% from 0.48+/-0.47 mmol/L (18.4+/-18.3 mg/dL) to 0.59+/-0.54 mmol/L (22.9+/-21.0 mg/dL) after 2 months (P<.05), and it was reduced by 30.6% to 0.41+/-0.51 mmol/L (15.9+/-20.1 mg/dL)(P<.005) with therapy with conjugated equine estrogen (Premarin). The Lp(a) lipoprotein levels were not changed in the group of women who underwent a TAH and unilateral salpingo-oophorectomy. In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the high density lipoprotein cholesterol level showed a trend of increase after 2 months from 1.45+/-0.48 mmol/L (56.1+/-18.5 mg/dL) to 1.58+/-0.309 mmol/L (61.2+/-15.1 mg/dL) without statistical significance, and it revealed a significant elevation to 1.76+/-0.43 mmol/L (68.2+/-16.8 mg/dL) with therapy with conjugated equine estrogen (Premarin) compared with that of the basal level (P<.05). CONCLUSIONS: tHE Lp(a) lipoprotein levels appear to be closely associated with female sex hormones. This association might play a pivotal role in postmenopausal increases of atherosclerotic diseases and cardioprotective effect of estrogen in postmenopausal women.

Effect of a novel saponin adjuvant derived from Quillaja saponaria on the immune response to recombinant hepatitis B surface antigen.

Adjuvant activity of saponins extracted from the South American tree Quillaja saponaria has been demonstrated with many antigens. Recently, four saponin fractions (designated as QS-7, QS-17, QS-18, and QS-21) with adjuvant activity were purified by reverse phase chromatography. In particular, efficacy of the less toxic QS-21 fraction has been demonstrated with several recombinant viral antigens including HIV gp120. Here, we report a novel saponin fraction (designated as QS-L1) derived from Quillaja saponaria. Unlike previously identified saponins, QS-L1 had a different chemical structure and showed adjuvant activity only when administered in the presence of alum-precipitated antigen. Interestingly, the QS-L1 greatly increased not only a humoral immune response but also cellular immune response to recombinant hepatitis B virus surface antigen (HBsAg). Furthermore, QS-L1 showed lower toxicity in vivo and in vitro than the previously identified saponin fraction, QS-21. Finally, we examined the chemical structure of the QS-L1 using mass spectroscopic analysis, carbohydrate composition analysis and NMR spectroscopic analysis. Thus, our results indicated that this novel QS-L1 saponin fraction had several desirable properties required for an effective adjuvant.

A double-blind, randomised- placebo, controlled, parallel group, multicentre, flexible-dose escalation study to assess the efficacy and safety of sildenafil administered as required to male outpatients with erectile dysfunction in Korea.

The efficacy and safety of sildenafil was evaluated in a randomiSed, double-blind, placebo-controlled, flexible-dose study in Korean men aged 28-78 y with erectile dysfunction (ED) of broad-spectrum aetiology and more than 6 months duration. A total of 133 patients were randomised at six centres in Korea to receive either sildenafil (50 mg initially, increased if necessary to l00 mg or decreased to 25 mg depending on efficacy and tolerance) (n=66) or matching placebo (n=67) taken on an 'as needed' basis l h prior to anticipated sexual activity for a period of 8 weeks. At the end of this time, the primary efficacy variables relating to the achievement and maintenance of erections sufficient for sexual intercourse, and the secondary efficacy variables, which included: (1) the five separate domains of sexual functioning of the International Index of Erectile Function (IIEF) scale, (2) the percentage of successful intercourse attempts, and (3) a global assessment of erections, were all statistically significantly improved by sildenafil in comparison with placebo (P&<0.0001). Treatment-related adverse events occurred in 56.1% of patients receiving sildenafil and 20.9% receiving placebo. The most common adverse events with sildenafil were vasodilatation (flushing), headache and abnormalities in colour vision (31.8, 22.7 and 6.1% of patients, respectively), and most were mild in nature. The efficacy and safety of sildenafil in this population of Korean men appears similar to that reported in other studies in western populations.

Lithium pretreatment reduces brain injury after intracerebral hemorrhage in rats.

OBJECTIVE: In addition to the mood-stabilizing effects of lithium in patients with bipolar disorder, recent in vitro and in vivo studies in rodents increasingly implicate that lithium may be useful for treating acute cerebral ischemia, neuroinflammatory conditions, and chronic neurodegenerative diseases. However, whether lithium has a protective effect against hemorrhagic stroke is yet unknown. To test this possibility, we attempted to determine lithium's effect on experimental intracerebral hemorrhage (ICH). METHODS: We treated adult rats with either lithium (2 mEq/kg) or saline for 3 days before inducing ICH via a stereotaxic infusion of collagenase into the left basal ganglia. Hematoma volumes, hemispheric swelling, long-term hemispheric atrophy, microglial activation, cell death, cyclooxygenase-2 expression, and behavioral outcomes were assessed. RESULTS: Per behavioral tests 2 days after ICH, the lithium-treated group recovered better than did the saline-treated group. Three days after ICH, the hematoma volumes did not differ between the groups, but hemispheric swelling was less in the lithium-treated group. Forty-two days after ICH, hemispheric atrophy was less in the lithium-treated group. Lithium reduced cell death, cyclooxygenase-2 expression, and reactive microglia in the perihematomal regions. CONCLUSION: The present study shows that lithium, via anti-inflammation, reduces the perihematomal cell death, which is associated with sensorimotor recovery after experimental ICH.

Paradoxical longevity in obese patients with intracerebral hemorrhage.

BACKGROUND: The paradoxical phenomenon of relative longevity among obese patients with established diseases has been reported for various disease conditions. The authors sought to investigate whether the obesity paradox also applies to intracerebral hemorrhage (ICH) survivors. METHODS: A total of 1,604 patients with ICH from 33 centers with nationwide coverage were prospectively enrolled to this cohort between October 2002 and March 2004. Baseline information including body mass index (BMI) was collected at admission, and mortality status was ascertained from the governmental mortality archive on December 2006. Associations between obesity and 30-day mortality or long-term risk of death were analyzed. RESULTS: Among the 1,356 patients with ICH included, the 30-day mortality rate was 7.2% and the long-term mortality rate was 26.9% after a mean follow-up of 33.6 ± 15.5 months. Neither BMI nor obesity status were associated with 30-day mortality after ICH. However, BMI was independently associated with a lower risk of long-term mortality (hazard ratio [HR] 0.91 per 1-kg/m(2) increase; 95% confidence interval [CI] 0.87-0.95). As compared with patients of normal weight, underweight subjects had a higher risk of death (HR 1.64; 95% CI 1.11-2.40), and conversely, overweight (HR 0.69; 95% CI 0.49-0.96) or obese (HR 0.61; 95% CI 0.43-0.88) subjects showed a lower risk of post-ICH death. CONCLUSION: In our study, obesity was associated with a lower risk of long-term death but not with 30-day mortality after ICH. Thus, it may be considered that an obesity status in a patient with ICH be treated as an indication of metabolic reservoir capacity and an increased likelihood of survival.

Increased serum alkaline phosphatase as a predictor of long-term mortality after stroke.

BACKGROUND: Although the critical role of alkaline phosphatase in bone mineralization is clearly understood, the potentially adverse effect of high alkaline phosphatase levels on the cardiovascular system was only recently suggested. In this study, we hypothesized that increased levels of serum alkaline phosphatase may be associated with poor outcome after stroke in terms of mortality. METHODS: We prospectively included patients with acute stroke admitted consecutively to our hospital, from October 2002 to September 2008. A total of 2,029 patients were selected for the analyses. In the analyses of mortality, the patients were divided by baseline measurements into quintiles of alkaline phosphatase levels (<57, 57-69, 70-81, 82-97, >97 IU/L). RESULTS: In the Cox proportional hazard models, compared with the first alkaline phosphatase quintile, adjusted hazard ratios of the third, fourth, and fifth quintiles for all-cause death were 1.67 (95% confidence interval 1.12-2.49), 1.79 (1.20-2.67), and 2.83 (1.95-4.10). When we divided the patients into ischemic and hemorrhagic stroke, the association was also significant for both subtypes of stroke. In terms of vascular death, compared to the first alkaline phosphatase quintile, the adjusted hazard ratios of the fourth and fifth quintiles of alkaline phosphatase were 1.81 (95% confidence interval 1.14-2.86) and 2.78 (1.87-4.15). CONCLUSION: Our study demonstrated that increased serum levels of alkaline phosphatase are an independent predictor of all-cause and vascular death after either ischemic or hemorrhagic stroke.

White matter lesions and poor outcome after intracerebral hemorrhage: a nationwide cohort study.

BACKGROUND: The ability to predict poor outcome is important for patient care and treatment decision-making in cases of intracerebral hemorrhage (ICH). Previous studies have included relatively brief follow-up periods and small numbers of patients, and are limited in terms of considerations regarding individual brain vulnerabilities. METHODS: The authors prospectively enrolled 1,321 ICH patients nationwide from 33 hospitals. Clinical, laboratory, and imaging variables, including white matter lesions (WMLs), were collected at admission. Immediate outcome after ICH was measured using total Glasgow Coma Scale (GCS) score at admission, early outcome using 30-day mortality, and long-term outcome using relative risk for mortality. The vital status of included patients was ascertained on December 31, 2006, using Korean National Death Certificates (mean follow-up, 32.6 months). RESULTS: Of the 1,321 ICH patients included, 352 (27.8%) presented with a moderate GCS score (8.5-12.4) and 249 (19.7%) with a severe GCS score (

Simian virus 40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm.

Little or no simian virus 40 (SV40) late mRNA accumulates in the cytoplasm when the primary transcript lacks an excisable intervening sequence. To begin to understand why, we analyzed the synthesis, processing, transport, and stability of SV40 late transcripts accumulated in the nucleus and cytoplasm of monkey cells cotransfected with the DNAs of wild-type and mutants of SV40 lacking precisely various introns. The data from these experiments indicated that (i) the presence of excisable intervening sequences in SV40 late transcripts is necessary for efficient accumulation in the cytoplasm of any of the SV40 late RNA species and (ii) SV40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm but not in stability in the cytoplasm. We hypothesize that SV40 late transcripts need to be processed via a pathway that couples stabilization of the primary transcript within the nucleus, excision of intervening sequences, proper 5'- and 3'-end formation, and transport to the cytoplasm.

Assembly of hepatitis delta virus particles.

Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV). Since the RNA genome of HDV can replicate in cultured cells in the absence of HBV, it has been suggested that the only helper function of HBV is to supply HBV coat proteins in the assembly process of HDV particles. To examine the factors involved in such virion assembly, we transiently cotransfected cells with various hepadnavirus constructs and cDNAs of HDV and analyzed the particles released into the medium. We report that the HDV genomic RNA and the delta antigen can be packaged by coat proteins of either HBV or the related hepadnavirus woodchuck hepatitis virus (WHV). Among the three co-carboxy-terminal coat proteins of WHV, the smallest form was sufficient to package the HDV genome; even in the absence of HDV RNA, the delta antigen could be packaged by this WHV coat protein. Also, of the two co-amino-terminal forms of the delta antigen, only the larger form was essential for packaging.

Presentation of the hydrophilic domains of hepatitis C viral E2 envelope glycoprotein on hepatitis B surface antigen particles.

Subviral particles of hepatitis B virus have been used to present foreign epitopes. We attempted to present the hydrophilic domains of E2 envelope protein of hepatitis C virus (HCV) as a fusion protein with hepatitis B virus surface antigen (HBsAg). The five hydrophilic domains of HCV E2 antigen were inserted into HBsAg such that the inserted hydrophilic domains were presented on the outer surface of HBV subviral particles. In addition, a fusion encoding the hypervariable region (HVR) of E2 antigen was also made. Cell lysate and culture medium were analyzed for the synthesis and secretion of the fusion proteins by immunoprecipitation with polyclonal anti-HBsAg antibody using recombinant vaccinia virus system. The results showed that the fusion proteins containing these six E2 domains were made in the cell, but only two out of six fusion proteins were secreted into culture medium. Further, cesium chloride density gradient analysis and electron microscopy revealed that these fusions were secreted into culture media as particles. It will be of interest to test immunogenicity of the HBsAg fusion particles containing the HCV E2 domains in animal model.

Evidence that the 5'-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA.

Hepatitis B virus (HBV) replicates by reverse transcription of an RNA intermediate, the pregenomic RNA. The first step of HBV genome replication is the encapsidation of the pregenomic RNA encoding the encapsidation signal, termed epsilon, into the core particles, which is preceded by recognition and binding of HBV DNA polymerase to epsilon. The pregenomic RNA contains two identical epsilon elements due to its terminal redundancy: one near the 5' end and another near the 3' end. Despite the fact that both epsilon elements have an identical sequence, only the 5' epsilon, but not the 3' epsilon, is functional for encapsidation. To understand the molecular nature of this position effect, we made a series of lacZ RNA expression plasmids which contain the epsilon element at various positions from the 5' end of the transcripts. Following transfection, the lacZ RNAs in cytoplasmic core particles were measured by RNase protection assay for encapsidation. The results indicated that the lacZ RNAs with epsilon positioned up to 65 nucleotides from the 5' end were encapsidated, whereas the lacZ RNAs with epsilon positioned further downstream were not. Interestingly, the cap-free lacZ RNA transcribed by T7 RNA polymerase was not encapsidated, implying that the 5' cap structure is required for encapsidation of the pregenomic RNA. We hypothesized that HBV DNA polymerase must somehow recognize the cap structure and/or its associated factors, as well as the 5' epsilon, for encapsidation to occur.