Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC.

Gelsolin is a Ca2+-dependent actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility through modulation of the actin network. Gelsolin was also recently suggested to be involved in the regulation of apoptosis: human gelsolin (hGsn) has anti-apoptotic activity, whereas mouse gelsolin (mGsn) exerts either proapoptotic or anti-apoptotic activity depending on different cell types. Here, we studied the basis of anti-apoptotic activity of hGsn. We showed that both endogenous and overexpressed hGsn has anti-apoptotic activity, that depends on its C-terminal half. We also found that hGsn and its C-terminal half but not mGsn could prevent apoptotic mitochondrial changes such as Apsi loss and cytochrome c release in isolated mitochondria to a similar extent as Bcl-xL, indicating that hGsn targets the mitochondria to prevent apoptosis via its C-terminal half. In the same way as anti-apoptotic Bcl-xL, which we recently found to prevent apoptotic mitochondrial changes by binding and closing the voltage-dependent anion channel (VDAC), hGsn and its C-terminal half inhibited the activity of VDAC on liposomes through direct binding in a Ca2+-dependent manner. These results suggest that hGsn inhibits apoptosis by blocking mitochondrial VDAC activity.

Dose-dependent doxycycline-mediated adrenocorticotropic hormone secretion from encapsulated Tet-on proopiomelanocortin Neuro2A cells in the subarachnoid space.

We previously reported that polymer-encapsulated mouse neuroblastoma cells that are capable of secreting beta-endorphin may reduce pain sensitivity in rats after capsule implantation into the cerebrospinal fluid (CSF)-filled subarachnoid space of the spinal cord. The neuroblastoma cells carry the proopiomelanocortin (POMC) gene that encodes the precursor of adrenocorticotropic hormone (ACTH) and beta-endorphin. To control the expression of these hormones in the present study, a promoter that is inducible by administration of tetracycline derivatives such as doxycycline (Dox) was linked to the POMC gene. Encapsulated cells in the CSF space of rats stimulated by four intraperitoneal doses of Dox responded with ACTH expression as determined in a subsequence 36-hr in vitro incubation. The amount of ACTH released was dependent on the in vivo Dox dose. These findings indicate that gene expression in xenogeneic cells in the CSF space can be manipulated by injection of a relatively innocuous drug, and suggest that this system may be applicable to cell transplantation therapy in patients with central nervous system diseases that require temporary control of ligand delivery.

Lipid peroxidation as a possible cause of benzoyl peroxide toxicity in rabbit dental pulp--a microsomal lipid peroxidation in vitro.

The toxicity of composite resin on rabbit dental pulp was investigated biochemically. A microsomal fraction of rabbit dental pulp was incubated with each of the components of composite resins, and the formation of peroxide was determined by the thiobarbituric acid reaction. Benzoyl peroxide (BPO), the most widely used catalyst, was the most effective on peroxidation, but monomers were not. Cations such as Cu2+ or Fe2+ were required for acceleration of this reaction. Authentic polyunsaturated fatty acids and phospholipids were extensively converted into their peroxides by BPO, but amino acids and carbohydrates were not. Among the active oxygens, hydroxyl radicals were thought to be responsible for BPO-dependent peroxidation. The results presented in this paper indicate that the lipid portion of the cells may be attacked by hydroxyl radicals produced by BPO and copper or iron. Therefore, BPO is considered to be the major factor responsible for the toxicity of composite resins.

[Experience with polyurethane intraurethral stents in aged patients].

A double Malecot-like 16F, polyurethane intraurethral catheter (IUC) was inserted 39 times in 25 patients between 68 and 91 years old (mean +/- SD: 77.5 +/- 5.5 years). Most of them were unfit for surgery because of severe illness or advanced age. All patients except 1 had either an indwelling catheter or a residual urine volume > 50ml at their first visit. The remaining one suffered from prostatic cancer and complained of pollakisuria and urinary incontinence. His symptoms were controlled well after ICU insertion and anticholinergic therapy. Twenty-nine insertions in 17 patients were considered successful, and the patients with successfully inserted stents voided without residual urine. Nine of 21 patients needed to take anticholinergic drugs. The mean duration of IUC use was 113 days in successful cases. In 9 out of 17 patients with an indwelling IUC for more than 112 days, the catheter became blocked by stones or clots. Therefore, we consider that the device should be changed after 110-120 days. Ten insertions failed for the following reasons: malposition, inappropriate IUC length, spontaneous migration to the bladder in patients with a short prostatic urethra, urinary retention due to underactive detrusor, and total incontinence and bleeding from prostatic cancer.

[Effects of long-term administration of oxybutynin hydrochloride (KL007) for the treatment of neurogenic bladder and unstable bladder].

The efficacy and safety of long-term administration of oxybutynin hydrochloride in patients with neurogenic bladder and unstable bladder, who complained of urinary frequency, urgency and incontinence, and whose bladder was proved to be uninhibited, reflex, and/or low compliant, were studied at the multi-center hospitals, and the following results were obtained. This study comprised 149 cases but 8 of them were excluded because of the incomplete protocol. Thus, 141 cases (104 neurogenic bladder patients, 33 unstable bladder patients and 4 others) were investigated. The daily dosage of Oxybutynin hydrochloride ranged from 1 to 18 mg, and averaged at 6.6 mg. Since a small daily dosage of 3 mg was administered in 32 cases to examine the minimum effective dosage of the drug, the low average dosage possibly resulted. The clinical optimal dosage seemed to be more than 6.6 mg. The average period of administration in all cases was 161.7 days (1-336 days), and the drug was discontinued in 46 cases (33.6%) on average 62.1 days (1-141 days). The rate of global improvement by this drug estimated at the time of completion was found to be 65.6% with excellent and good and 87.8% with excellent, good and fair. The efficacy of this drug was stable and not decreased during the long-term test period. As to the objective findings studied before, during and after the drug administration, cystometric bladder capacity was significantly increased both first desire to void and the total capacity. Voided volume and residual urine were also increased, but there was no change in the rate of residual urine. Side effects were observed in 37 (26.2%) of 141 cases, and mainly gastrointestinal signs such as dry mouth and constipation. Urological signs such as dysuria and urinary retention were experienced in 8 cases. Regarding the findings of laboratory tests, there were no abnormality except for small changes of some items in normal range. Six children who were younger than 15 years old were subjected to this study. The results of evaluation were similar to those obtained on adults and no side effects were observed. From these findings, oxybutynin hydrochloride is considered an effective and useful drug in patients suffering from neurogenic bladder and unstable bladder with over active bladder condition.

Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.

During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L). Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.

Study on hypomineralized areas in hamster molars.

Hypomineralized area (HMA) scores were determined for hamster molars during tooth maturation and fluoride treatment. It was found that the molars were maturing until the animals were 45 days old, but from 45 to 74 days of age the HMA scores remained constant. Fluoride treatment decreased the HMA score. The group with the lowest HMA score was fed a cariogenic diet, inoculated with Streptococcus mutans and received topical fluoride treatment.

Dye penetration of the smear layer and fluoride application to the dentin surface.

A smear layer is formed after cavity or root canal preparation. The aim of the present study was to reinforce the dental surface in order to prevent the invasion of foreign irritants, by treating the smear layer with fluoride. Dentinal samples whose surfaces had been washed with water after the formation of a smear layer, and corresponding samples without washing, were examined by the dye penetration test, and the results were compared. Although there was no significant difference between the two groups of samples, dye penetration was suppressed by about 30% in washed samples, whereas the suppression was 20% in unwashed samples. When washed samples were treated with 1.0% SnF2, 10.0% SnF2, 7.5% Na2PO2F, 15% Na2PO2F, APF, 10 mM In(NO3)3, 100 mM TiF3, 50 mM TiF3, or 10 mM TiF3, samples washed after treatment with 1.0% SnF2 showed a dye penetration suppression of about 60% as a whole, in comparison with samples having no smear layer. Hardly any suppression of dye penetration was observed after treatment with other fluorides.

Effect of smear layer removal agent and/or fluoride on the surface of dentin.

A study was conducted to observe the effectiveness of an EDTA-based agent, Tubulicid Red, and a polyacrylic acid-based agent, Dentin Conditioner, for removal of the smear layer from a prepared dentin surface, with or without a fluoride dentin reinforcing agent, using the dye penetration test and scanning electron microscopy (SEM). Bovine mandibular first and second incisors were used. After removal of the enamel, the smear layer on the dentin surface was treated with 38% H3PO4, Tubulicid Red, Dentin Conditioner, 1% NaF, 1% SnF2, Tubulicid Red (including 1% SnF2), Dentin Conditioner (including 1% NaF) and Dentin Conditioner (including 1% SnF2). In the dye penetration test, Dentin Conditioner (including 1% SnF2) was the most effective agent for preventing dye penetration. SEM evaluation of the dentin surface after treatment with the smear layer removal agents and/or fluorides showed that the smear layer was removed by H3PO4 and Dentin Conditioner. However, dentinal plugs remained after treatment with Dentin Conditioner alone. The other agents left some layers on the dentin surface.

Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

Manganese inhibits benzoyl peroxide/copper-dependent lipid peroxidation in the microsomal fraction of rabbit dental pulp.

1. Of all the cations tested only manganese ion inhibited the benzoyl peroxide/Cu2+-dependent formation of thiobarbituric acid (TBA)-reactive substance. 2. Ki of Mn2+ for the formation of TBA-reactive substance was 5.0 microM. 3. The inhibitory manner of manganese was non competitive against copper.