Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.     

Gait Variability before Surgery and at Discharge in Patients Who Undergo Total Knee Arthroplasty: A Cohort Study

This study aimed to determine gait ability at hospital discharge in patients undergoing total knee arthroplasty (TKA) as an indicator of the risk of falling. Fifty-seven patients undergoing primary TKA for knee osteoarthritis participated in this study. Gait variability measured with accelerometers and physical function including knee range of motion (ROM), quadriceps strength, walking speed, and the Timed Up and Go (TUG) test were evaluated preoperatively and at discharge from the hospital (1 month before and 5 days after surgery). All patients were discharged directly home at 5 days after surgery. Knee flexion of ROM, quadriceps strength, walking speed, and the TUG test results were significantly worse at hospital discharge than preoperatively (p < 0.001). However, gait variability was not significantly different before and after TKA. This result indicated that patients following TKA surgery could walk at hospital discharge as stably as preoperatively regardless of the decrease in physical function, including knee ROM, quadriceps strength, and gait speed after surgery.     

Impact of the Distance from the Stent Edge to the Residual Plaque on Edge Restenosis following Everolimus-Eluting Stent Implantation

Objectives

This study aimed to assess the relation between stent edge restenosis (SER) and the distance from the stent edge to the residual plaque using quantitative intravascular ultrasound.

Background

Although percutaneous coronary intervention with drug-eluting stents has improved SER rates, determining an appropriate stent edge landing zone can be challenging in cases of diffuse plaque lesions. It is known that edge vascular response can occur within 2 mm from the edge of a bare metal stent, but the distance to the adjacent plaque has not been evaluated for drug-eluting stents.

Methods

A total of 97 proximal residual plaque lesions (plaque burden [PB] >40%) treated with everolimus-eluting stents were retrospectively evaluated to determine the distance from the stent edge to the residual plaque.

Results

The SER group had significantly higher PB (59.1 ± 6.1% vs. 51.9 ± 9.1% for non-SER; P = 0.04). Higher PB was associated with SER, with the cutoff value of 54.74% determined using receiver operating characteristic (ROC) curve analysis. At this cutoff value of PB, the distance from the stent edge to the lesion was significantly associated with SER (odds ratio = 2.05, P = 0.035). The corresponding area under the ROC curve was 0.725, and the cutoff distance value for predicting SER was 1.0 mm.

Conclusion

An interval less than 1 mm from the proximal stent edge to the nearest point with the determined PB cutoff value of 54.74% was significantly associated with SER in patients with residual plaque lesions.     

Alterations in hepatitis B virus nucleotide sequences in a chronic virus carrier from immunotolerant to immunoactive phase

Factors involved in transition from the immunotolerant to immunoactive phase in chronic hepatitis B virus (HBV) infection remain unclear. We investigated viral mutations occurring during transition and elucidated their virological and immunological significance. Full-length HBV DNA sequences were serially determined in a chronic HBV carrier from the immunotolerant to immunoactive phase. Viral replicative competence was examined by transfection analysis. HBV-specific CD8⁺ T cell response was evaluated by coculture of CD8⁺ T cells with autologous dendritic cells followed by interferon-γ Elispot assay. Eleven point mutations and two deletions appeared around the onset of the immunoactive phase. Viral replicative competence declined significantly after the onset of active hepatitis. Examination of the CD8⁺ T cell response against two putative T-cell epitopes, which contained substituted amino acids from the immunotolerant to immunoactive phase, showed that mutant HBV epitopes gave a lesser T cell response than wild-type HBV ones. In summary, point mutations and deletions may occur prior to or concurrent with the onset of the immunoactive phase during chronic HBV infection. These mutations may result in a significant decrease in both viral replicative competence and HBV-specific CD8⁺ T cell response, suggesting a possible adaptation for the maintenance of viral persistence.     

Protective role of macrophages in noninflammatory lung injury caused by selective ablation of alveolar epithelial type II Cells

Macrophages have a wide variety of activities and it is largely unknown how the diverse phenotypes of macrophages contribute to pathological conditions in the different types of tissue injury in vivo. In this study we established a novel animal model of acute respiratory distress syndrome caused by the dysfunction of alveolar epithelial type II (AE2) cells and examined the roles of alveolar macrophages in the acute lung injury. The human diphtheria toxin (DT) receptor (DTR), heparin-binding epidermal growth factor-like growth factor (HB-EGF), was expressed under the control of the lysozyme M (LysM) gene promoter in the mice. When DT was administrated to the mice they suffered from acute lung injury and died within 4 days. Immunohistochemical examination revealed that AE2 cells as well as alveolar macrophages were deleted via apoptosis in the mice treated with DT. Consistent with the deletion of AE2 cells, the amount of surfactant proteins in bronchoalveolar lavage fluid was greatly reduced in the DT-treated transgenic mice. When bone marrow from wild-type mice was transplanted into irradiated LysM-DTR mice, the alveolar macrophages became resistant to DT but the mice still suffered from acute lung injury by DT administration. Compared with the mice in which both AE2 cells and macrophages were deleted by DT administration, the DT-treated LysM-DTR mice with DT-resistant macrophages showed less severe lung injury with a reduced amount of hepatocyte growth factor in bronchoalveolar lavage fluid. These results indicate that macrophages play a protective role in noninflammatory lung injury caused by the selective ablation of AE2 cells.     

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.     

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.     

Calpain 6 is involved in microtubule stabilization and cytoskeletal organization

The calpains are a family of Ca(2+)-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated alpha-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated alpha-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.     

Rotenoids and flavonoids with anti-invasion of HT1080, anti-proliferation of U937, and differentiation-inducing activity in HL-60 from Erycibe expansa

Principal rotenoids (deguelin, tephrosin, rotenone, and 12a-hydroxyrotenone) (3-30microM) isolated from the stems of Erycibe expansa significantly inhibited invasion of human fibrosarcoma HT1080 cells through Matrigel-coated filters and release of proMMPs-2 and 9. In addition, deguelin and tephrosin showed differentiation-inducing activity in human promyelocytic leukemia HL-60 cells. Furthermore, effects of various constituents isolated from the ethyl acetate-soluble fraction on proliferation of human leukemia U937 cells were examined. As a result, most of isoflavones and several flavans as well as rotenoids showed moderate or substantial anti-proliferative activities.     

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg²⁺- and Gd³⁺-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg²⁺ and Gd³⁺. The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca²⁺ was removed or Gd³⁺ was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca²⁺ influx pathway, it plays an important role in cell volume regulation. regulatory volume decrease