Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.     

Hybrid MR interventional imaging system: combined MR and angiography suites with single interactive table. Feasibility study in vascular liver tumor procedures

Our objective was to evaluate the feasibility of a hybrid system consisting of a high-field MR and fully equipped digital subtraction angiography (DSA) unit for MR-guided vascular interventions. In a newly built hybrid system, consisting of a high-field MRI and a fully equipped DSA unit, elective interventional hybrid procedures were performed. Between May 2000 and November 2001, 30 patients with liver tumors underwent MR-guided chemoembolization using the hybrid system. During the intervention accurate catheter position was monitored with real-time and dynamic MR imaging. Elective hybrid interventional vascular procedures were performed successfully in 23 patients with liver metastases and hepatocellular carcinoma ( n=7). Patients could be transferred between the MRI and angiographic units on a carbon fiber tabletop within 10 s. Initial clinical trials demonstrated that in the chemoembolization of primary and secondary liver tumors the hybrid approach resulted in a change of catheter position in 40% of procedures. In combining high-field MR system and a fully equipped interventional vascular angiographic unit as backup, this hybrid system improves the therapeutic capabilities of interventional vascular procedures in the liver.     

基于分形和小波的混合图像压缩

从近年来受人瞩目的分形和小波图像压缩方法出发,分析了两种图像压缩方法的特点及利弊。运用小波域及DCT域下的分形压缩图像方法,提高了编码速度,得到较高的压缩比和信噪比,但重构图像质量还存在块状效应。为此,提出了几种方法结合起来的综合方法,进一步提高图像的质量,在压缩比和信噪比之间取得良好的折衷。     

Alcohol impairs protein synthesis and degradation in cultured skeletal muscle cells

BACKGROUND: Acute and chronic alcohol intoxication decreases skeletal muscle protein synthesis under in vivo conditions. We investigated whether ethanol (EtOH) and its major metabolites, acetaldehyde and acetate, can directly modulate protein balance under in vitro conditions. METHODS: Human myocytes were incubated with different doses of EtOH for varying periods of time (i.e., 4-72 hr). Alternatively, cells were incubated with acetaldehyde, acetate, insulin, insulin-like growth factor-I (IGF-I), or with a combination of EtOH plus insulin or IGF-I. Rates of protein synthesis or degradation were determined by 35S-methionine/cysteine incorporation into or release from cellular protein. RESULTS: A significant, 15% to 20%, decrease in basal protein synthesis was observed after 24 hr, but not at earlier time points, in response to 80 mM EtOH. Incubation of myocytes for 72 hr decreased synthesis in cells incubated with EtOH ranging between 60 and 120 mM. The ability of IGF-I or insulin to stimulate protein synthesis was impaired by 30% and 60%, respectively, in cells incubated with 80 mM EtOH for 72 hr. Exposure of cells to 200 microM acetaldehyde or 5 mM Na-acetate also decreased basal protein synthesis. In contrast, neither EtOH, acetaldehyde, nor acetate altered the basal rate of protein degradation. However, EtOH completely impaired the ability of insulin and IGF-I to inhibit proteolysis. Finally, EtOH did not impair IGF-I receptor autophosphorylation, but inhibited the ability of insulin to phosphorylate its own receptor. EtOH also did not alter the number of insulin or IGF-I receptors or the formation of insulin/IGF-I hybrid receptors. CONCLUSIONS: We have demonstrated that EtOH can directly inhibit muscle protein synthesis under in vitro conditions. Neither EtOH nor its metabolites altered basal protein degradation, although EtOH did compromise the ability of both insulin and IGF-I to slow proteolysis. This impairment seems to be mediated by different defects in signal transduction.     

Therapeutic Effect of Ansamitocin Targeted to Tumor by a Bispecific Monoclonal Antibody

We have constructed a murine hybrid hybridoma that secretes a bispecific monoclonal antibody (mAb) by fusing a hybridoma secreting an anti-ansamitocins mAb with a hybridoma secreting an anti-human transferrin receptor (TfR) mAb that binds to human A431 epidermoid carcinoma cells. The bispecific mAb, reactive to both ansamitocins and TfR, was purified by a combination of hydrophobic column chromatography and hydroxyapatite high-performance liquid chromatography, and evaluated in in vivo experiments using human tumor cell-implanted nude mice. Ansamitocin P-3 targeted through one of the antigen combining sites of the bispecific mAb was potentially more effective in suppressing the growth of established A431 tumor xenografts implanted on nude mice than unconjugated ansamitocin P-3 or the immunoconjugate of ansamitocin P-3 and monospecific anti-ansamitocins antibody, and the targeted ansamitocin P-3 finally eradicated the tumor mass. The bispecific mAb also played an important role in reducing such undesirable side-effects of ansamitocin P-3 as the loss of body weight, the damage to liver functions and the decrease in the number of white blood cells.     

A volume-sensitive Cl conductance in a mouse neuroblastoma × rat dorsal root ganglion cell line (F11)

Whole cell currents were recorded in F11 cells, a mouse neuroblastoma (NG18TG2) × rat DRG hybrid cell line, using pipette and bath solutions intended to isolate any chloride conductance pathways. When recording with a pipette solution which was 40 mmol·kg⁻¹ hypotonic to the bath solution, all cells showed a transient rise in input conductance which peaked 5.3±0.4 min after breaking into the cell and returned to the basal state 11.7±1.2 min later. At the peak of the effect, cell conductance had increased approximately sixfold. The use of short (300 ms) duration voltage steps at the peak of the conductance increase evoked whole-cell currents which were time-independent and had an outwardly rectifying current/voltage relationship. Ion substitution experiments showed that the whole-cell currents were carried by chloride ions and that the anion selectivity sequence of the conductance was I > Br > Cl > F > acetate. The stilbene derivative 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) caused a reversible, 51% inhibition of the chloride currents. In cells which had already undergone this transient rise in conductance, whole-cell currents with identical properties could be activated by changing to a very hypotonic bath solution. Coincident with current activation, this manoeuvre caused a visible swelling of the cell. The increase in conductance and the cell swelling were both reversed by returning to the normal bath solution. In contrast, when a very hypotonic pipette solution was used, little or no increase in cell conductance was observed. These data suggest that the F11 cell line possesses a volume-activated chloride conductance which can be controlled by manipulating the relative osmolarity of the bath and pipette solutions.     

Marginal and internal adaptation of Class II ormocer and hybrid resin composite restorations before and after load cycling

To overcome the shortcomings of the conventional composite restorative materials, ormocer materials have been introduced over the past few years. The purpose of this study was to evaluate the marginal and internal adaptation of two ormocer restorative systems (Admira, Voco and Definite, Degussa) compared to a hybrid composite one (TPH Spectrum, Dentsply/ DeTrey), before and after load cycling in Class II restorations. Standardized Class II restorations with cervical margins on enamel were divided into three groups (n=16). Teeth of each group were filled with one of the restoratives tested and its respective bonding agent. Each group was divided into two equal subgroups. The marginal and internal adaptation of the first subgroup was evaluated after 7-day water storage at room temperature and of the second after cyclic loading in a mastication simulator (1.2×10⁶ cycles, 49 N, 1.6 Hz). The occlusal and cervical marginal evaluation was conducted by videomicroscope and ranked as excellent and not excellent. One thin section (150 m), in mesial-distal direction, of each restoration, was examined under metallographic microscope to determine the quality of internal adaptation. The occlusal and cervical adaptation of both ormocer restorative systems was similar and clearly worse compared with the hybrid composite restorative one before as well as after load cycling. Concerning internal adaptation, no gap-free ormocer restorations were detected, whereas all Spectrum restorations presented perfect adaptation. The bonding agents of the ormocers formed layers with unacceptable features (pores, fractures) whereas that of the hybrid composite achieved perfect bonding layer even after loading. The rheological characteristics of the bonding agents of the ormocer restorative systems are proposed to be responsible for their inferior marginal and internal quality in Class II restorations compared with the hybrid composite one.     

Human papillomavirus DNA detection and histological findings in women referred for atypical glandular cells or adenocarcinoma in situ in their Pap smears

OBJECTIVE: To evaluate the association between high-risk human papillomavirus (HPV) DNA detection and histological diagnosis in women referred for atypical glandular cells (AGC) or adenocarcinoma in situ (AIS) at Pap smear. METHODS: In this cross-sectional study, 146 women referred for AGC (124), AGC with high-grade squamous intraepithelial lesion (HSIL) (15), or AIS (7) were tested for HPV DNA using Hybrid Capture II (HC II). All women underwent colposcopic examination, and cervical biopsy was performed for 95 patients. Fifty-one women referred due to AGC with normal colposcopy and normal second Pap smear were scheduled for control visits every 4 months. RESULTS: The overall prevalence of HPV DNA was 38%. HPV DNA was detected in 93% of the women with HSIL associated with AGC and in 71% of women with AIS Pap smear, being significantly higher when compared with the prevalence (29%) in women with AGC alone. Forty-five women (30.8%) had clinically significant histological lesions (CIN 2 or worse). High-risk HPV DNA was detected in only 16% of the women without significant abnormalities in biopsy, in contrast to 96% of those who had CIN 2 or CIN 3 and 75% of women with AIS. Eighty-five percent of women with invasive cervical carcinoma (squamous or adenocarcinoma) tested positive for HPV DNA. HPV DNA detection was significantly associated with histological diagnosis of CIN 2 or worse, with an odds ratio (OR) = 51.8 (95% CI 14.3-199.9). CONCLUSION: HPV DNA detection was strongly associated with the severity of cervical lesion (CIN 2 or worse) in women referred for AGC or AIS in their Pap smear. These data implicate the use of HPV testing in triage of women with AGC Pap smears.     

Characterization of three rabbit oral papillomavirus oncogenes

Rabbit oral papillomavirus (ROPV) induces warts in mucosal tissues, and represents a useful model for understanding host-virus interactions that are reflected in mucosal/HPV infections. ROPV induces benign papillomas that regress in 100% of infected rabbits. We previously reported the complete genome sequence of ROPV. However, the oncogenic potential of this virus is unknown because of immunologically mediated regression. The purpose of this study was to characterize the transforming proteins of E6, E7, and E8 genes of ROPV. E6, E7, and E8 genes of ROPV were cloned into the expression vector PCR3. Two hybrid CRPV-ROPV E6 genes were also constructed and tested together with the three wild-type ROPV genes. Each construct was transfected into NIH3T3 cells and stable transfected cell lines were established. Transforming properties of ROPV E6, E7, and E8 were tested via anchorage-independent growth of cells in agar plates and tumor growth in athymic mice. Cells with ROPV E6, E7, or E8 formed colonies in agar and tumors in athymic mice. These observations suggest that ROPV E6, E7, and E8 are oncogenic.     

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.