Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm)

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH(m)). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.     

Alterations of antiproliferative effects of serum obtained from patients with acute cerebral infarction treated with a radical scavenger, edaravone, with or without amlodipine using an in vitro cultured basilar artery smooth muscle cells

The guinea-pig basilar artery smooth muscle cell (GBa-SM3) culture system in the Dulbecco's modified Eagle's medium for 3 days serves as a useful in vitro model for assessing antiproliferative effects of various therapeutic agents on vessels. With use of this system we studied whether human serum obtained from patients with acute cerebral infarction (n = 16) would have a proliferative effect on vessels and whether an administration of a free radical scavenger, edaravone, with or without amlodipine would elicit antiproliferative effects. The control serum was obtained from 3 healthy human subjects. Time courses of the cell growth and survival were measured colorimetrically by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrzolium bromide (MTT) test. The stimulatory effect on the proliferation of GBa-SM3 cells of patients' serum obtained immediately after infarction was significantly (p < 0.05) greater than those obtained from the same patients after the treatment of edaravone for 2 weeks. In addition, the serum obtained from the patients treated by edaravone and amlodipine (n = 7) showed a significantly (p < 0.05) greater antiproliferative effect than that obtained from those treated by edaravone (n = 9). In conclusion, edaravone may have a clinically beneficial antiproliferative effect on vascular smooth muscle cells. Co-administration of amlodipine, possessing an antioxidative calcium channel blocker, with edaravone may be a promising combination to patients with acute cerebral infarction. Further controlled clinical trials with a large number of patients should be warranted.     

Effects of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, 3-hydroxy-3-methyl glutaryl (HMG) CoA reductase inhibitors, amlodipine and epalrestat on cultured basilar artery smooth muscle cell proliferation

Proliferation of vascular smooth muscle cells (VSMC) stimulated by oxidative stresses and reactive oxygen species (ROS) may play a pivotal role in the pathogenesis of atherosclerosis. Antiatherosclerotic effects of angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, HMG CoA reductase inhibitors, calcium channel blocker and epalrestat were studied with an in vitro guinea-pig basilar artery smooth muscle cell (GBa-SM3) culture system over 3 days incubated with 0 to 10% of fetal bovine serum. Results demonstrated that simvastatin (0.1 mM), fluvastatin (0.3 mM), amlodipine (0.2 mM) and epalrestat (1 mM) elicited significant (p < 0.05 or 0.01) antiproliferative effects, whereas losartan (1 mM), valsartan (1 mM), enalapril (0.1 mM), captopril (1 mM), trandolapril (0.01 mM), pravastatin (0.7 mM) did not. In conclusion, the present in vitro VSMC culture system may serve as a comprehensive screening method for pleiotropic effects of commonly used therapeutic agents.     

A case of the development of cavum vergae after head trauma

We report a case of the development of cavum septi pellucidi and cavum Vergae after head trauma in a 29-year-old female patient. After the accident, cavum septi pellucidi and cavum Vergae gradually expanded. Using magnetic resonance imaging, we followed up the patient for 33 months after the head trauma. Preoperatively, metrizamide CT cisternography was performed in order to investigate CSF flow between the cavum septi pellucidi/cavum Vergae and the subarachnoid space. Using neuroendoscopy, we succeeded in opening the wall of the cavum septi pellucidi via anterior horn of the right lateral ventricle. Cystography was performed during the operation. The volume of the cavum septi pellucidi/cavum Vergae decreased remarkably. In this case, our CSF flow studies revealed that CSF flowed into the posterior part of the cavum Vergae from the third ventricle and did not flow backward to the third ventricle. Therefore, we considered that the development of cavum septi pellucidi/cavum Vergae was related to a one-way valve mechanism between the posterior part of the cavum Vergae and the third ventricle.     

The sharing of target-epitopes between human anti-ABO hemagglutinating and anti-Pig (xeno) endothelium or anti-Pig thyroglobulin antibodies

To select congenial pairs between donor-pig and recipient-human for the future xenotransplantation, the levels of xeno-IgM natural antibodies (NAb) were analyzed in healthy subjects and hemodialysis patients by ELISA tests, which target swine-derived crude endothelial cells (P16N) or proteins (thyroglobulin; TG). The total IgM concentration was lower in hemodialysis patients than in healthy subjects, but there was no difference in IgM NAb titer between the two groups. Individuals with non-B blood types (A, O) exibited significantly higher IgM NAb titer compared with those with B blood types (B, AB). A blood type individuals showed higher killing activity against P16N than those with B, AB or O types with a statistical significance. Sera from A blood type, after being absorbed with red blood cells (RBC) from B blood type, decreased their IgM titer against TG to the level of sera from B blood type. Meanwhile, sera from A blood type significantly decreased hemagglutinin titer against B-RBC after passage through a TG-coated affinity column. We conclude that human anti-B-RBC and anti-Pig xeno NAb have certain common binding epitopes, which might be a branched B carbohydrate structure.     

Coeruleotrigeminal suppression of nociceptive sensorimotor function during inflammation in the craniofacial region of the rat

Descending action from the locus coeruleus (LC) on the trigeminal sensorimotor function was evaluated in a rat model of oral-facial inflammation. For the induction of oral-facial inflammation, mustard oil (20% solution in 20microl mineral oil) was injected into the region of the temporomandibular joint (TMJ). One week before testing, rats received bilateral lesions of the LC using a cathodal current. The electromyogram (EMG) threshold, which is the threshold intensity for the onset of EMG activity of the masseter muscle evoked by pressure on the TMJ region, was used in the present study as an indicator of the trigeminal sensorimotor function. Following mustard oil injection, in the LC-lesioned rats, EMG thresholds significantly decreased at 30min, which lasted up to 240min. In contrast, EMG thresholds in the LC-intact rats returned to the level before injection after 180min. Systemic naloxone (1.3mg/kg, i.v.) produced a further decrease of EMG thresholds in both the LC-intact and LC-lesioned rats. Under the existence of naloxone, EMG thresholds in the LC-lesioned rats were significantly lower than those of the LC-intact rats. These results suggest that oral-facial inflammation activates the coeruleotrigeminal modulating system and that an action of this system is independent of the opioid depressive mechanism.     

Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli

BACKGROUND: We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions. RESULTS: The present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 degrees C. Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division. CONCLUSIONS: A single cluster of SeqA-bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies.     

Adult Leigh syndrome with mitochondrial DNA mutation at 8993

Adult onset Leigh syndrome with a nucleotide (nt) 8993 mutation in mitochondrial (mt) DNA is reported. A 43-year-old woman with a 6-year-history of insulin-resistant diabetes mellitus developed muscular weakness, intractable nausea and vomiting, and anemia. These were followed vertigo, blindness, and deafness with nystagmus. Magnetic resonance imaging (MRI) revealed abnormal high intensities in the bilateral medial regions of the thalamus and periaqueductal gray matters. Autopsy disclosed well-demarcated necrotizing lesions with prominent capillaries in the areas detected by MRI, which were sufficiently diagnostic for Leigh syndrome. MtDNA analysis performed on DNAs extracted from formalin-fixed tissues including liver, heart, brain, muscle, kidney and pancreas showed a T→G mutation at nt 8993. This is the first case of adult Leigh syndrome demonstrating on mtDNA mutations. Received: 24 August 1998 / Revised, accepted: 21 October 1998     

The effect of the bisphosphonate ibandronate on breast cancer metastasis to visceral organs

Bisphosphonate (BPs), specific inhibitors of osteoclastic bone resorption, are widely used therapeutic agents for bone metastases in breast cancer patients. Nevertheless, the effects of BPs on visceral metastases are controversial. Here we specifically studied the effects of the BP ibandronate on visceral metastases of breast cancer using two animal models. In the first set of experiments, we examined the effects of ibandronate on lung metastasis using 4T1 mouse mammary tumor that developed pulmonary and bone metastases following orthotopic inoculation in syngeneic female Balb/c mice. In the second set of experiments, we examined the effects of ibandronate on adrenal metastasis using a clone of the MDA-MB-231 (MDA-231) human breast cancer (MDA-231AD cells) that developed adrenal and bone metastases following intracardiac inoculation in female nude mice. These breast cancer cells were stably transfected with a firefly luciferase cDNA to facilitate quantification of the metastatic tumor burden in visceral organs. Ibandronate (4 µg/day, sc, daily) was given either after metastases were established (therapeutic administration) or at the time of tumor cell inoculation (preventative administration). In both models with each protocol, ibandronate reproducibly reduced bone metastases, establishing that BPs are effective pharmacological agents for the treatment of bone metastases in breast cancer. In the 4T1 model, neither the preventative nor therapeutic administration of ibandronate caused any effects on lung metastases. In the MDA-231 model, the preventative administration of ibandronate significantly increased adrenal metastases. However, no increase in the adrenal metastases was observed when an anti-cancer agent doxorubicin was co-administered. Therapeutic administration of ibandronate showed no effects on the adrenal metastases. Our results suggest that BPs cause no adverse effects on visceral metastases when administered in the manners that breast cancer patients usually receive.     

Laryngeal and respiratory responses to tracheal irritation at different depths of enflurane anesthesia in humans

The effect of three different depths of enflurane anesthesia (1.0, 1.4, and 1.8 MAC) upon laryngeal and respiratory responses to tracheal instillation of distilled water in nine female patients in whom a double-cuffed endotracheal tube had been inserted was investigated. The laryngeal responses were monitored by measuring the pressure in the saline-filled cuff positioned within the larynx, and the respiratory responses were monitored by measuring ventilatory flow and tracheal airway pressure. Increases in laryngeal cuff pressure in response to tracheal irritation were 19.7 +/- 4.5 cmH2O (mean +/- SD) at 1.0 MAC, 13.9 +/- 3.6 cmH2O at 1.4 MAC, and 7.6 +/- 1.8 cmH2O at 1.8 MAC, respectively (P less than 0.01 for anesthetic dose). At 1.0 MAC of enflurane anesthesia, tracheal instillation of saline caused immediate laryngeal constriction and all components of the tracheal response, such as apnea, expiration reflex, cough reflex, and spasmodic panting. At 1.4 and 1.8 MAC, the same stimulation caused only apnea and constriction of the larynx in the majority of patients. These results indicate that changes in depth of anesthesia can modify the laryngeal and respiratory responses to tracheal irritation. The close association of laryngeal and respiratory responses may be an integral part of the defensive reflex synergism.