Zonal necrosis prevented by transduction of the artificial anti-death FNK protein.

Protection of cells from necrosis would be important for many medical applications. Here, we show protein transduction domain (PTD)-FNK therapeutics based on protein transduction to prevent necrosis and acute hepatic injury with zonal death induced by carbon tetrachloride (CCl4). PTD-FNK is a fusion protein comprising the HIV/Tat PTD and FNK, a gain-of-function mutant of anti-apoptotic Bcl-x(L). PTD-FNK protected hepatoma HepG2 from necrotic death induced by CCl4, and additionally, increased the apoptotic population among cells treated with CCl4. A concomitant treatment with a pan-caspase inhibitor Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), which alone could not prevent the necrosis, protected these cells from the apoptosis. When pre-injected intraperitoneally, PTD-FNK markedly reduced zonal liver necrosis caused by CCl4. Moreover, injection of PTD-FNK accompanied by Z-VAD-FMK suppressed necrotic injury even after CCl4 administration. These results suggest that PTD-FNK has great potential for clinical applications to prevent cell death, whether from apoptosis or necrosis, and organ failure.

Lipoma and opthalmoplegia in mitochondrial diabetes associated with small heteroplasmy level of 3243 tRNA(Leu(UUR)) mutation.

We report a patient with mitochondrial diabetes mellitus associated with the A3243G mutation (MDM3243). The patient is a 77-year man with diabetes. At age 68, he noticed diplopia, due to superior rectus muscle palsy of the right eye. At age 70, he noticed lipoma on the right arm. The pathology of his muscle revealed some ragged-red fibers, and focal cytochrome c oxidase deficiency. Hence, he may have a pathogenetic mechanism in common with CPEO (chronic progressive external ophthalmoplegia) or mitochondria-related autoimmune disorder associated with mononeuropathy. He had the rate of 0.102% for heteroplasmy of 3243 mitochondrial DNA mutation in leukocytes. This case's heteroplasmy level is the smallest among the reported cases of MDM3243 in the literature. 3243 mitochondrial DNA mutation is known to induce a lack of uridine-modification in tRNA(Leu(UUR)) at the first letter of the anticodon, with which the third letter of the codon pairs, and decline of the pairing of the anticodon of tRNA with the codon of mRNA, suggesting the termination of polypeptide-elongation to generate premature proteins. Therefore, we speculate that these premature proteins may accumulate overtime, thereby affecting cells in target organs.

Accumulation of somatic mutation in mitochondrial DNA extracted from peripheral blood cells in diabetic patients.

AIMS/HYPOTHESIS: A point mutation of mitochondrial DNA at nucleotide number 3243 A to G is responsible for both the major genetic aetiologies of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and mitochondrial diabetes. Otherwise, this mutation is also reported to occur as an acquired somatic mutation, possibly due to oxidative stress. Since diabetes can cause severe oxidative stress, we hypothesize that the accumulation of the somatic 3243 A to G mutation in mitochondrial DNA can be accelerated by diabetes. METHODS: DNA was extracted from blood samples of 290 non-diabetic healthy subjects (age 20-60) including 98 newborn infants and from 383 patients with Type II (non-insulin-dependent) diabetes mellitus (age 18-80). The extent of somatic 3243 A to G mutation to total mitochondrial DNA was detected by real-time PCR using the TaqMan Probe. RESULTS: Whereas the level of the 3243 A to G mutation was negligible in the newborn group, it was increased in healthy subjects who were 20 to 29 and 41 to 60 years of age, suggesting that this mutation was somatic. In the diabetic patients the mutation rate increased along with age and the duration of diabetes. In the middle-aged group (age 41-60), the 3243 A to G mutation accumulates fourfold higher in the diabetic patients than the healthy subjects. Moreover, multiple regression analysis showed that the most critical factor associated with this mutation in diabetic patients was the duration of diabetes. CONCLUSION/INTERPRETATION: Diabetes accelerates the accumulation of the somatic 3243 A to G mutation in mitochondrial DNA, which can accelerate the ageing process. This somatic mutation could possibly be a new marker for estimating the duration of diabetes.

Localization of Bcl-xbeta in the developing and adult rat central nervous system.

bcl-xbeta is a novel apoptosis-regulating member of the bcl-x family that has recently been isolated from rats and mice. To explore the functional role of Bcl-xbeta, we raised a monoclonal antibody against rat Bcl-xbeta protein and investigated the cellular localization of the molecule in the rat CNS. Immunohistochemistry revealed that, in the fetal and neonatal stages, Bcl-xbeta was intensively and widely expressed in the CNS. Many neurons in the diencephalon and brain stem showed intense cytoplasmic labeling. The immunoreactivity decreased during the postnatal development and reached to the level of adulthood by P14. In the adult brain and spinal cord, labeling was restricted to specific types of neurons and distributed throughout their somata and dendrites. Weak immunoreactivity was present in many CNS regions such as the cerebral cortex, hippocampal dentate gyrus, caudate-putamen, globus pallidus, thalamus, locus ceruleus, pontine nuclei, inferior olive, reticular formation, cerebellar cortex and spinal anterior horn. Amygdaloid nuclei and hippocampal CA1 to CA3 sectors showed restricted expression of Bcl-xbeta in a subset of neurons. Neuronal labeling was almost undetectable in several regions, including the piriform cortex, hypothalamus, posterior column nuclei and spinal posterior horn. These results suggest that Bcl-xbeta plays an important role throughout the CNS in developing stage and may regulate the apoptosis of postnatal CNS neurons.

Bacterial cell death induced by human pro-apoptotic Bax is blocked by an RNase E mutant that functions in an anti-oxidant pathway.

BACKGROUND: Bax is a member of the Bcl-2 family and induces apoptosis of mammalian cells. We have shown that a trace amount of human Bax induces the cell death of Escherichia coli, accompanied by damage to DNA, and that the region of Bax which is lethal to E. coli is also responsible for apoptosis-inducing activity in the mammalian cells. RESULTS: We isolated a Bax-resistant mutant from E. coli cells that survive in the presence of paraquat, a generator of superoxide, by screening a library constructed from the random insertion of a transposon. Psb1 (paraquat-resistant, suppressor of Bax-1) mutant had a Tn 10 transposon inserted in the rne gene of E. coli, splitting the RNase E gene (rne) into N- and C-terminal halves. The introduction of the truncated 5' end of rne specifically enhanced resistance to paraquat, prevented cell death induced by Bax and decreased the intracellular H2O2 concentration. The region responsible for the paraquat- and Bax-resistance was not the catalytic site for the endoribonuclease activity of RNase E. CONCLUSIONS: The N-terminal region of the RNase E protein inhibits bacterial death induced by human Bax as well as paraquat through a unique mechanism that is distinct from RNA digestion. This study implies that the protection of bacterial death induced by Bax is associated with an anti-oxidant pathway and that a mutant RNase E has a novel function as an anti-oxidant.

HLA DPB1*0201 gene confers disease susceptibility in japanese with childhood onset type I diabetes, independent of HLA-DR and DQ genotypes.

HLA is an important etiologic genetic factor in Type I diabetes and specific HLA-class II genes are closely related to the onset of the disease. Many differences in the patterns of susceptible and resistant DRB1, DQA1, and DQB1 genes have been observed among various ethnic groups. We have previously shown that DRB1*0405, DRB1*0901 and DQA1*0301-DQB1*0302 were the major susceptible alleles or haplotype to Type I diabetes while DR-DQ haplotype studies suggested the important role of DR and DQ alleles in susceptibility and resistance in Japanese patients. Based on the analysis of 90 Japanese patients with childhood onset Type I diabetes and 136 unrelated healthy Japanese controls by polymerase chain reaction-restriction fragment polymorphism method (PCR-RFLP), we report here the association of Type I diabetes with DPB1*0201 (relative risk = 2.29; Pc = 0.027) in this population. Comparison of linkage disequilibrium patterns between patients and controls showed that the significantly high prevalence of DPB1*0201 among patients cannot be attributed simply to linkage disequilibrium with susceptible DRB1 alleles and DQA1-DQB1 haplotypes. Our results suggest that in addition to alleles at the DRB1, DQA1, DQB1 loci, polymorphism at DPB1 locus also influences the risk of Type I diabetes.

Melanoma in situ of the penis.

Melanoma of the penis is rare and the prognosis is very poor. We report a case of melanoma in situ localized on the penile shaft. Melanoma in situ of the penis is extremely rare. We emphasize that early diagnosis of melanoma in situ will improve the prognosis of melanoma of the penis.

Evaluation of local intra-arterial fibrinolytic therapy for acute middle cerebral artery occlusion.

SUMMARY: Fibrinolytic therapy for acute ischaemic stroke has been investigated in several clinical trials, with various protocols. This retrospective study was undertaken to evaluate the efficacy and limitation of local intra-arterial fibrinolytic therapy using urokinase (UK) in patients with acute middle cerebral artery occlusion. Fifty patients were treated with local intra-arterial fibrinolytic therapy within six hours after onset of symptoms. The median National Institutes of Health Stroke Scale (NIHSS) score was 17 (range, 6 to 28).Two hundred and forty thousand IU of UK was administered through a microcatheter for 20 minutes. When arterial recanalization was not achieved, a second or third infusion was performed. Maximum dosage of UK was 0.96 x 106 IU. Recanalization efficacy was evaluated at the end of fibrinolytic therapy and intracranial haemorrhage was assessed within 24 hours. Clinical outcome was evaluated three months after ictus with modified Rankin scale (RS). Thirty-nine patients (78%) obtained recanalization. Twenty-nine of 39 (74%) showed clinical improvement just after treatment. On the other hand, only 18% patients (2/11) who did not recanalize demonstrated improvement. Twenty-five of 50 (50%) patients recovered to RS score 0 or 1, however, only 28% of patients (5/18) with proximal M1 occlusion obtained good outcome and 39% of them (7/18) died. The mean time interval from onset to treatment did not affect outcome. The overall incidence of haemorrhagic event (HE) within 24 hours was 36%, however, 78% of patients with proximal M1 occlusion showed HE. Only one patient with HE clinically deteriorated. In conclusion, local intra-arterial fibrinolytic therapy could be a safe and effective method for acute middle cerebral artery occlusion, however, indication of this therapy for patients with proximal M1 occlusion should be carefully decided.

Enhanced embryonic nonmuscle myosin heavy chain isoform and matrix metalloproteinase expression in aortic abdominal aneurysm with rapid progression.

Abdominal aortic aneurysms (AAAs) are characterized by structural deterioration of aortic wall leading to progressive dilatation. The histopathological changes in AAAs are particularly evident within the elastic media, which is normally comprised mainly of vascular smooth muscle cells (SMCs). There are vascular myosin heavy chain (MHC) isoforms; SM2 is specifically expressed in differentiated SMCs and SMemb is a nonmuscle-type MHC abundantly expressed in SMCs of the fetal aorta with an immature phenotype. Although AAA altered expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), pathophysiological role of SMC phenotypic modulation in the AAA progression remains uncertain. To determine whether phenotypic modulation in vascular SMCs contributes to arterial medial degeneration, we examined MHC expression in SMCs of AAA. Aortic specimens were obtained from patients with slowly progressed AAA (n = 12) and rapidly progressed AAA (n = 5), and compared with normal aortic tissue (n = 3). Immunohistochemical staining was performed for detection of SMemb, SM2, MMP (types 2 and 9) and TIMP (types 1 and 2). Faint SMemb and abundant SM2 were observed in normal aorta, while the balance shifted to SMemb predominance in AAAs. Compared with slowly progressed AAA tissue, rapidly expanded AAA tissue demonstrated marked increases in SMemb expression with suppressed SM2. Predominant SMemb expression indicates presence of phenotypic modulated SMCs and enhanced MMP; while abundant TIMP was seen in mature SMCs expressing SM2. SMemb expression is markedly increased in AAA with MMP enhancement, and a significant imbalance between SMemb and SM2 results in rapid progression of AAA.

Embolization of cerebral arteriovenous malformations achieved with polyvinyl alcohol particles: angiographic reappearance and complications.

BACKGROUND AND PURPOSE: The appropriate choice of embolic materials with respect to the permanency of obliterated nidi after embolization and complications related to the procedure is essential for safe and effective embolization of cerebral arteriovenous malformations (AVMs). Our purpose was to ascertain the recanalization and complication rates after AVM treatment with polyvinyl alcohol (PVA) particles. METHODS: Between 1988 and 1994, 36 AVMs were embolized with PVA particles at our institution. Follow-up angiographic findings and occurrence of complications during the embolization procedures were analyzed retrospectively. RESULTS: Complete obliteration of the nidus immediately after embolization was achieved in five patients, and 80% to 99% obliteration was attained in 12 patients. Fifty-one follow-up angiographic examinations were performed 1 week to 60 months (mean, 7 months) after embolization in 31 patients. An increase in nidal size was seen on 15 follow-up angiograms (29%) and a decrease was seen in seven (14%). In 28 of the 51 angiograms obtained more than 1 month after follow up (mean, 13 months), 12 (43%) showed AVM enlargement. In four (80%) of five cases of complete obliteration, nidi reappeared on follow-up angiograms. Hemorrhagic complications occurred in three cases and ischemic ones in seven. One patient (3%) died and five (14%) suffered persistent neurologic deficits. CONCLUSION: Embolization with PVA particles can produce significant volume reduction in AVM nidal size, but recanalization is a distinct possibility.

Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer's disease.

To determine whether pathogenic mutations in mtDNA are involved in phenotypic expression of Alzheimer's disease (AD), the transfer of mtDNA from elderly patients with AD into mtDNA-less (rho0) HeLa cells was carried out by fusion of platelets or synaptosomal fractions of autopsied brain tissues with rho0 HeLa cells. The results showed that mtDNA in postmortem brain tissue survives for a long time without degradation and could be rescued in rho0 HeLa cells. Next, the cybrid clones repopulated with exogenously imported mtDNA from patients with AD were used for examination of respiratory enzyme activity and transfer of mtDNA with the pathogenic mutations that induce mitochondrial dysfunction. The presence of the mutated mtDNA was restricted to brain tissues and their cybrid clones that formed with synaptosomes as mtDNA donors, whereas no cybrid clones that isolated with platelets as mtDNA donors had detectable mutated mtDNA. However, biochemical analyses showed that all cybrid clones with mtDNA imported from platelets or brain tissues of patients with AD restored mitochondrial respiration activity to almost the same levels as those of cybrid clones with mtDNA from age-matched normal controls, suggesting functional integrity of mtDNA in both platelets and brain tissues of elderly patients with AD. These observations warrant the reassessment of the conventional concept that the accumulation of pathogenic mutations in mtDNA throughout the aging process is responsible for the decrease of mitochondrial respiration capacity with age and with the development of age-associated neurodegenerative diseases.

A trace amount of the human pro-apoptotic factor Bax induces bacterial death accompanied by damage of DNA.

An amount of human pro-apoptotic Bax as low as 0.01% of total protein was sufficient to cause cell death in Escherichia coli. The bacterial cell death was examined using a viable bacteria-specific fluorescence indicator system and loss of colony formation ability. Co-expression of anti-apoptotic Bcl-xL showed a modest inhibitory effect on the cell death caused by Bax. The trace amount of Bax elongated E. coli and accumulated monounsaturated fatty acids, suggesting an unusual metabolism of redox in the host. In fact, an increase of KCN-dependent O2 consumption accompanied the expression of Bax. At the same time, a fluorescent pH indicator showed the apparent accumulation of protons outside the cell, suggesting that the membrane is intact. Bax increased the level of superoxide anion as measured by the expression of superoxide-dependent promoter. Nicked DNA was significantly generated, and the frequency of mutations resistant to rifampicin was increased by 30-fold, depending upon the expression of Bax. It is proposed that trace amounts of Bax increase oxygen consumption, triggering generation of superoxide, which affects DNA, leading to bacterial death.

Pore formation domain of human pro-apoptotic Bax induces mammalian apoptosis as well as bacterial death without antagonizing anti-apoptotic factors.

A trace amount of the pro-apoptotic factor human Bax was sufficient to kill host Escherichia coli (Asoh, S., Nishimaki, K., Nanbu-Wakao, R., and Ohta, S., submitted). The region of Bax lethal to E. coli cells was determined by introducing truncated human bax mutant genes. A peptide corresponding to amino acid residues 115 to 144 of Bax was the smallest peptide capable of inducing cell death of E. coli. A truncated bax gene (Bax112-192) containing the region lethal to E. coli was then introduced into a murine promyeloid cell line, FDC-P1. Constitutively expressed Bax112-192 induced apoptosis as judged by decrease of transfectants surviving and DNA fragmentation. These results indicate that Bax112-192 contains the region directly responsible for mammalian apoptosis as well as bacterial death. Flow cytometric analysis by FITC-Annexin V showed that the transfectant cells expressing Bax112-192 or native Bax became apoptotic even without external stimuli. The apoptotic population in the cells expressing Bax112-192 was not decreased by co-expression of Bcl-2 or Bcl-XL, while Bcl-2 or Bcl-XL suppressed apoptosis in the cells expressing native Bax. Therefore, Bax induces apoptosis by its own activity without blocking the anti-apoptotic activity involved in Bcl-2 or Bcl-XL.

Non-contact specular microscopic observation for early response of corneal endothelium after contact lens wear.

PURPOSE: Contact lenses induce short- and long-term corneal endothelial changes, including endothelial blebs. The endothelial bleb is a reversible, short-term response found on the corneal endothelium after lens wear. We used non-contact specular microscopy to observe endothelial blebs occurring with different types of contact lenses. METHODS: We observed the time course, frequency, and location of endothelial blebs in 11 eyes of 9 contact lens wearing patients. Eight types of contact lenses with various oxygen transmissibilities (Dk/L) were used in the study. RESULTS: Blebs were observed in the central cornea in patients with polymethylmethacrylate and rigid gas permeable contact lenses, while blebs in the central to mid-peripheral cornea were seen in patients wearing soft contact lenses. CONCLUSIONS: Although the number of blebs vary by individual patient, there appears to be an inverse correlation between the number of blebs and the Dk/L of the contact lens.