A mechanical coil insertion system for endovascular coil embolization of intracranial aneurysms.

Like other fields of medicine, robotics and mechanization might be introduced into endovascular coil embolization of intracranial aneurysms for effective treatment. We have already reported that coil insertion force could be smaller and more stable when the coil delivery wire is driven mechanically at a constant speed. Another background is the difficulty in synchronizing operators' minds and hands when two operators control the microcatheter and the coil respectively. We have therefore developed a mechanical coil insertion system enabling a single operator to insert coils at a fixed speed while controlling the microcatheter. Using our new system, the operator manipulated the microcatheter with both hands and drove the coil using foot switches simultaneously. A delivery wire force sensor previously reported was used concurrently, allowing the operator to detect excessive stress on the wire. In vitro coil embolization was performed using three methods: simple mechanical advance of the coil; simple mechanical advance of the coil with microcatheter control; and driving (forward and backward) of the coil using foot switches in addition to microcatheter control. The system worked without any problems, and did not interfere with any procedures. In experimental coil embolization, delivery wire control using the foot switches as well as microcatheter manipulation helped to achieve successful insertion of coils. This system could offer the possibility of developing safer and more efficient coil embolization. Although we aim at total mechanization and automation of procedures in the future, microcatheter manipulation and synchronized delivery wire control are still indispensable using this system.

Cell type-specific transactivation of the VCAM-1 promoter through an NF-kappa B enhancer motif.

Cytokine activation of vascular cell adhesion molecule-1 (VCAM-1) gene expression by endothelial cells is an important feature in a variety of vascular inflammatory responses. Cytokines transcriptionally activate the VCAM-1 promoter in endothelial cells at least in part through two closely linked NF-kappa B enhancer motifs, kappa L-kappa R (positions -77 and -63). However, cytokine activation of the dimeric NF-kappa B transcriptional factor (p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1 gene expression exhibits a cell type-specific pattern of expression. Tumor necrosis factor-alpha markedly transactivated a transiently transfected minimal kappa L-kappa R motif-driven VCAM-1 promoter, p85VCAMCAT, in passaged human vascular endothelial cells but not in the human epithelial cell line, HeLa suggesting that cell type-specific factors may function through the kappa L-kappa R motif. Both cell types exhibited similar inductions of NF-kappa DNA binding activity and transcriptional activity. However, co-transfection of HeLa cells with p65 and p50 expression vectors demonstrated that the minimal VCAM-1 promoter was effectively transactivated by p65 alone but that additional co-expression of p50 blocked this activity. Furthermore, cytokine activation of the minimal VCAM-1 promoter in HeLa cells was recovered by inhibition of p50 expression using antisense oligonucleotide. These studies suggest that the NF-kappa B(p50+p65 heterodimer) does not support transactivation of the VCAM-1 promoter with the p50 subunit potentially playing a significant inhibitory role in suppressing cytokine activation of VCAM-1. In addition, p65 associated transcriptional factors other than NF-kappa B may serve as positive, cytokine-inducible, cell type-specific regulators of VCAM-1 gene expression.

Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells.

Oxidative stress and expression of the vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells are early features in the pathogenesis of atherosclerosis and other inflammatory diseases. Regulation of VCAM-1 gene expression may be coupled to oxidative stress through specific reduction-oxidation (redox) sensitive transcriptional or posttranscriptional regulatory factors. In cultured human umbilical vein endothelial (HUVE) cells, the cytokine interleukin 1 beta (IL-1 beta) activated VCAM-1 gene expression through a mechanism that was repressed approximately 90% by the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC). Furthermore, PDTC selectively inhibited the induction of VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1), mRNA and protein accumulation by the cytokine tumor necrosis factor-alpha (TNF alpha) as well as the noncytokines bacterial endotoxin lipopolysaccharide (LPS) and double-stranded RNA, poly(I:C) (PIC). PDTC also markedly attenuated TNF alpha induction of VCAM-1-mediated cellular adhesion. In a distinct pattern, PDTC partially inhibited E-selectin gene expression in response to TNF alpha but not to LPS, IL-1 beta, or PIC. TNF alpha and LPS-mediated transcriptional activation of the human VCAM-1 promoter through NF-kappa B-like DNA enhancer elements and associated NF-kappa B-like DNA binding proteins was inhibited by PDTC. These studies suggest a molecular linkage between an antioxidant sensitive transcriptional regulatory mechanism and VCAM-1 gene expression that expands on the notion of oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis.

ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.

Genistein arrests cell cycle progression at G2-M.

Genistein, an isoflavone, is a specific inhibitor of tyrosine kinase and topoisomerase II. However, its effect on cell growth is unknown. Therefore, we examined the effects of genistein on cell growth and cell cycle progression and compared its effects with other flavonoids. Genistein inhibited in a dose-dependent manner the growth of HGC-27 cells derived from human gastric cancer. Flow-cytometric analysis showed that genistein almost completely arrested the cell cycle progression at G2-M. This effect was reversible when genistein was removed from the culture medium. In contrast, other flavonoids such as flavone, luteolin, and the structurally similar daidzein arrested the cell cycle at G1. Consistent with the flow-cytometric analysis, microscopic observation showed that genistein did not increase the mitotic index, which supposes that genistein may arrest the cell cycle at G2 or early M. These results suggest that the G2-M arrest by genistein is a unique effect among flavonoids.

Bradycardia-induced abnormal QT prolongation in patients with complete atrioventricular block with torsades de pointes.

Fourteen patients with complete atrioventricular block with or without torsades de pointes (TdP) were included in this study. They were divided into 2 groups, 6 patients with TdP (TdP[+] group) and 8 patients without TdP (TdP[-] group). The patients were evaluated at 2 different periods, before (acute period) and after (chronic period) pacemaker implantation. In the acute period, the QRS and heart rate during the escape rhythm were not significantly different between the 2 groups; however, the QT and QTc intervals were significantly longer in the TdP(+) group than in the TdP(-) group: 753 +/- 57.5 vs 635 +/- 78.4 ms (p less than 0.01) and 585 +/- 44.8 vs 476 +/- 58.3 ms (p less than 0.01). In the chronic period (greater than 2 months after pacemaker implantation), we changed the pacemaker rate from 90 or 100 beats/min to 50 beats/min and examined the QT interval changes in relation to the heart rate. The QT interval in the TdP(+) group was significantly prolonged compared with the TdP(-) group when the pacing rate was decreased less than or equal to 60 beats/min: 551 +/- 40 vs 503 +/- 36 ms at 60 beats/min (p less than 0.05), and 700 +/- 46 vs 529 +/- 43 ms at 50 beats/min (p less than 0.001). Patients with complete atrioventricular block with TdP had a bradycardia-sensitive repolarization abnormality and this characteristic remained after pacemaker implantation. The critical heart rate that induced abnormal QT prolongation in the TdP(+) group was less than or equal to 60 beats/min.

Delta 12-prostaglandin J2 mimics heat shock in inducing cell cycle arrest at G1 phase.

Using a human neuroblastoma cell line GOTO, the effects of delta 12-prostaglandin (PG) J2 on the modulation of cell cycle progression and protein synthesis were examined in comparison with those caused by heat shock (HS). delta 12-PGJ2 induced G1 arrest, the peak of which was obtained at 24 h and continued for 72 h. HS was found to induce G1 arrest earlier than delta 12-PGJ2. Furthermore, sequential HS could maintain G1 arrest. delta 12-PGJ2 induced the synthesis of several heat shock proteins (HSPs) in a manner similar to HS. Using immunoblot analysis, HSP72 was detected prior to inducing G1 arrest and accumulated during the subsequent 72h. The content of HSP72 induced by HS also correlated well with the induction, release, and maintenance of G1 arrest. In addition, both delta 12-PGJ2 and HS induced HSP72 mRNA and simultaneously suppressed N-myc mRNA expression. These results suggest that delta 12-PGJ2 and HS regulate cell cycle progression of GOTO cells via similar mechanisms.

Induction of heat shock proteins and their implication in protection against ethanol-induced damage in cultured guinea pig gastric mucosal cells.

The induction of heat shock proteins in cultured guinea pig gastric mucosal cells was investigated to assess their role in gastric cytoprotection. In response to sublethal heat stress at 43 degrees C for 1 hour, the cells synthesized a 72-kilodalton protein and increased the synthesis of 74- and 90-kilodalton proteins, which were detected using gel electrophoresis after [35S]methionine labeling of the cells. Immunoblot analysis indicated that the 72- and 74-kilodalton proteins were members of the heat shock protein 70 family. Northern blot analysis showed the induction of a 2.6-kilobase messenger RNA of heat shock protein 70 gene only with heat treatment. Furthermore, with heat treatment, there was significant reduction of damage after ethanol treatment. This reduction was blocked with a protein synthesis inhibitor, cycloheximide, and was associated with inhibition of synthesis of heat shock proteins. These results strongly suggest that synthesis of heat shock proteins plays an important role in the intracellular mechanism of gastric protection against ethanol.

Flavonoids inhibit the expression of heat shock proteins.

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.

N-myc suppression and cell cycle arrest at G1 phase by prostaglandins.

Effects of cyclopentenone prostaglandins, delta 12-prostaglandin (PG) J2 and PGA2 on the expression of N-myc in relation to the effects on cell cycle progression were investigated using human neuroblastoma cell line GOTO. Both PGs suppressed N-myc expression within several hours prior to inducing G1 arrest. The N-myc suppression with delta 12-PGJ2 was continued but with PGA2 it was gradually released, followed by the release of G1 arrest. These results suggest that delta 12-PGJ2 and PGA2 inhibit cell cycle progression in strong association with N-myc suppression and delta 12-PGJ2 is more potent and has a longer effect than PGA2.

Inhibitory effect of quercetin on the synthesis of a possibly cell-cycle-related 17-kDa protein, in human colon cancer cells.

Quercetin inhibits growth of COLO320 DM cells, derived from a human colon cancer. The inhibitory effect is partially reversible when quercetin is removed from the culture medium. Flow cytometric analysis has revealed that quercetin causes perturbation of the cell cycle, inducing a frozen cell-cycle pattern and a block at the G1/S boundary. The synthesis of a 17-kDa protein was specifically inhibited by the addition of quercetin, and recovered when the cells at the G1/S boundary progressed into S-phase after the removal of quercetin from the culture medium. Furthermore, using synchronized cells obtained by centrifugal elutriation, we have shown that the rate of synthesis of a 17-kDa protein was low in G1, and high in S-phase of the cell cycle. Thus, this protein appears to be cell-cycle-related.

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells.

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC50 value was 32-55 microM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 microM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.

Studies on age-related functional changes in regulatory T cells and B cells involved in the autoantibody production of MRL/MpJ- +/+ mice.

Age-related changes in anti-DNA autoantibody production of MRL/MpJ- +/+ mice were investigated. In lipopolysaccharide (LPS)-stimulated cultures, spleen cells of the mice showed an age-related, marked increase in the ability to produce IgG class of the autoantibody after the age of 12 months, while they showed a tendency to decrease with age in the production of IgM class of the autoantibody. Serum levels of anti-DNA autoantibodies rose markedly in the IgG autoantibody but not in the IgM autoantibody after 12 months of age, which is well consistent with the observation in the LPS-stimulated cultures. T cell-depleted spleen cells, however, showed only a small increase with age in the IgG autoantibody productive ability. These results suggest that the age-associated increase in the IgG autoantibody production in the mice is under T-cell control. Age-associated changes in suppressor capacity in spleen cells of the mice were also investigated. Suppressive activity of the cells stimulated by 2-day incubation with concanavalin A (Con A) showed a clear increase as the donor age advanced, when assayed on the LPS-stimulated anti-DNA autoantibody production in vitro. The results indicate that, in MRL/MpJ-+/+ mice, suppressor capacity does not decline with age and is not related as a cause to the autoantibody production.

Protection by cycloheximide against cytotoxicity induced by vincristine, colchicine, or delta 12-prostaglandin J2 on human osteosarcoma cells.

We examined the protective effects of cycloheximide against cytotoxicity induced by vincristine, colchicine, delta 12-prostaglandin J2, or other antitumor agents on the human osteosarcoma cell line, KSu. Vincristine at a concentration of 0.5 microgram/ml decreased the initial cell number to 34% during 4 days; however, when cycloheximide (0.5 to 10 micrograms/ml) was coexistent, the decrease of the cell number was suppressed and 68% of the initial cells remained viable at the maximum. Furthermore, 0.1 micrograms/ml of cycloheximide also reduced cytotoxicity of colchicine (0.1 to 5 microM) or delta 12-prostaglandin J2 (1 to 5 micrograms/ml) and reduced the cytotoxicity of 0.1 microgram/ml of doxorubicin or 1 micrograms/ml of mitomycin C, suggesting that protection by cycloheximide is shown against cytotoxicity of various types of antitumor agents even on human malignant cells. Next, protein synthesis was reduced to 52% of a control at 3 h by 0.1 micrograms/ml of cycloheximide, suggesting that protein synthesis inhibition precedes the protection. De novo protein synthesis analysis showed that vincristine (0.5 microgram/ml) does not induce any specific protein, whereas delta 12-prostaglandin J2 (3 or 4 micrograms/ml) induced Mr 70,000 and 90,000 proteins, and these were markedly inhibited by cycloheximide (0.1 microgram/ml). In a cell-cycle study, M-phase arrest by vincristine (0.5 microgram/ml) was inhibited in the presence of 0.1 microgram/ml of cycloheximide, suggesting that cell cycle arrest by cycloheximide may be important for protection. From these data, this protection by cycloheximide seems to be more general than expected before.