PQBP-1 increases vulnerability to low potassium stress and represses transcription in primary cerebellar neurons.

PQBP-1 is a polyglutamine tract binding protein implicated in transcription. We previously reported that PQBP-1 and mutant ataxin-1, product of the spinocerebellar atrophy type 1 (SCA1) causative gene, cooperatively induce cell death in culture cells. Simultaneously, we showed that mutant ataxin-1 promoted interaction between PQBP-1 and RNA polymerase II and enhanced repression of the basal transcription by PQBP-1. In this study, we have examined the effects of overexpression of PQBP-1 to the primary-cultured cerebellar neurons. Our results indicate that overexpression of PQBP-1 inhibits the basal transcription in cerebellar neurons and increases their vulnerability to low potassium conditions.

Increase of CD5(+) B cells during adolescence in female mice.

We examined physiological changes in CD5(+) B lymphocytes in mice associated with aging (from 1 or 5 to 50 weeks of age). The most predominant populations of lymphocytes among these mice were mainly CD5(-) B cells in the spleen and peritoneal cavity, and T cells in the thymus. However, in the spleen, CD5(+) B cells increased from 1 to 15 weeks, and then decreased with aging. In the thymus, CD5(+) B cells increased from 3 to 9 weeks of age, and subsequently became more predominant than CD5(-) B cells. In the peritoneal cavity, CD5(+) B cells increased from 5 to 9 weeks of age, became the most predominant population in lymphocytes at 7 to 9 weeks of age, and decreased with aging. The proportion of CD5(+) B cells in total B cells increased from 5 to 7 or 9 weeks of age, and then decreased with aging, with the highest proportion at 9 weeks of age in the spleen (15%), thymus (94%), and peritoneal cavity (54%). These findings indicate that CD5(+) B cells increase physiologically during mouse adolescence, and subsequently decrease with aging.

Transfection of fluorescent probed antisense compounds: L-cysteine derivatives of nucleic acid bases.

Antisense with L-cysteine derivative (CAS) can recognize DNA and forms the complementary duplex with DNA. So the properties of CAS in vitro and in vivo were examined in this study. CAS was resistant to proteinase K and stabilized RNA against RNase HI. Moreover using fluorescent CAS, the localization was observed by fluorescence microscope and confocal microscope. As a result, CASs were accumulated inside the nucleus in NG108-15.

Study about the inhibition of L-cysteine derivatives of nucleic acid bases in protein production.

Isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acids bases were prepared as antisense compounds. In past study, we investigated the properties of these compounds in vitro, and revealed that these compounds in vivo regulated the cell death presumably due to the inhibition of protein production. In this study, western and northern blots were carried out in order to reveal the mechanism of this inhibition for N-methyl-D-aspartate receptor in neuroblastoma x glioma hybrid NG108-15 cell line. In addition, we investigated the resistance of these compounds against cell extract and the metabolism. In conclusion, we proved that these compounds inhibited the protein production by antisense mechanism.

[Video assisted thoracic surgery for the treatment of catamenial pneumothorax].

Video assisted thoracic surgery (VATS) was applied in 3 cases of pneumothorax combined with pathologic changes in the diaphragm (two cases of catamenial pneumothorax and one case of suspected catamenial pneumothorax). Case 1, 39-year-old woman, was preoperatively diagnosed as catamenial pneumothorax in the right lung. Thoracoscope was inserted through the 5th intercostal anterior axillary line and the lesion with the pathologic changes in the central tendon of the diaphragm was incised and sutured with Endo-GIA and Endo-STAPELAR. Case 2, 42-year-old woman, was confirmed to have pathologic changes in the central tendon after insertion of thoracoscope through the 5th intercostal anterior axillary line. Minithoracotomy of 50 mm in size was added close to the center of the diaphragm and direct incision and suture of that part were performed. Case 3, 47-year-old woman, underwent thoracoscopy through the 5th intercostal mid-axillary line and bulla in the S2 interlobar surface was incised with Endo-GIA. In this case, the diaphragm was partially incised through additional minithoracotomy because some lesions were detected on that part. VATS can fully be carried out for pathologic changes in the diaphragm in catamenial pneumothorax. Since catamenial pneumothorax may be complicated with another pathologic changes in the diaphragm (Case 1) or in the visceral pleura (Case 3), the whole thoracic cavity, including diaphragm and visceral pleura, should be carefully observed under thoracoscopy. Application of minithoracotomy-associated thoracoscopic surgery is a useful method in the case to whom catamenial pneumothorax is definite or suspected.

Cytokines production of U5A2-13-positive T cells by stimulation with glycolipid alpha-galactosylceramide.

We have previously established and reported a novel monoclonal antibody (mAb), U5A2-13, which recognizes a phenotypically similar population of natural killer (NK)-like T cells. Using U5A2-13 mAb, we now describe the functional properties of U5A2-13(+) T cells in both NK1.1-positive or -negative mouse strains. Similar to NK1.1(+) T cells, hepatic U5A2-13(+) T cells of C57BL/6 (NK1.1(+) strain) mice, but not U5A2-13(-) T cells, could be induced to produce large amounts of IL-4 and IFN-gamma by stimulation with glycolipid alpha-galactosylceramide (alpha-GalCer) present on dendritic cells (DC) in a dose-dependent manner. The abundant production of these cytokines from U5A2-13(+) T cells of BALB/c (NK1.1(-) strain) mice is similar to that noted in C57BL/6 mice. Cytokine production by cultures stimulated with DC of beta2-microglobulin-deficient mice was significantly less than that of cultures stimulated with DC of intact mice. Overall, U5A2-13(+) T cells recognize alpha-GalCer presented by CD1d, indicating that U5A2-13(+) T cells can be used to analyze NK-like T cell function in various strains of mice.

The behavior and effect of isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases.

Isopoly(S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases were prepared as antisense compounds. These compounds in vitro have been found to form stable complex with oligo-DNA or RNA. This paper deals with effect of antisense compounds in vivo. The target in this paper is the sequence of the PSD-95 protein linked with NMDA receptor. Excess passing of calcium ions through the loss of the signal pathway without PSD-95 proteins caused by antisense compound. The cells detailing with L-cysteine derivatives showed the lowest percentage of 19.1%. The data were compared with that of phosphotioate antisense compound.

Regression and prevention of autochthonous tumors induced by 3-methylcholanthrene after injection of a T-cell receptor alpha /beta positive and CD4/CD8 double negative T-cells.

Both the therapeutic and preventative effects of a murine T-cell line, tMK-2, with T-cell receptor (TCR) alpha/beta positive and CD4-/8- double negative (DN) phenotype against autochthonously tumors induced by subcutaneous (s.c.) injection of 3-methylcholanthrene (MC) were examined. Complete regression of the tumor was observed when administration of tMK-2 cells was begun on tumors 5 mm in diameter. The tumor mass in five out of five mice was reduced in size after the administration of tMK-2 cells regardless of the routes of administration: s.c. injection of tMK-2 cells (5 x 10(7) cells) once a week around tumors, intraperitoneal (i.p.) injection (5 x 10(7) cells), or intravenous (i.v.) injection (1 x 10(7) cells). The tumors regressed to the status of a scar within 1 month of initial injection, and this status was maintained throughout the remainder of the 3 months period of tMK-2 cell injection. One month after discontinuation of tMK-2 cell administration, the diameter of the tumors had not increased regardless of the route of injection. The control groups consisted of either untreated mice, mice with i.v. injection of 1 microg of recombinant murine interleukin (IL)-12 once a week, or mice with s.c. injection of autologous splenocytes (5 x 10(7)) from BALB/c mice once a week. Continuous growth of tumors was observed in each group and all control mice died due to bleeding ulcerations of the tumors. Tumor development was effectively prevented when tMK-2 cells were administrated 1 week after the s.c. injection of MC. In the groups receiving s.c., i.p., and i.v. injection of tMK-2 cells, no MC-induced tumors developed, whereas four out of five of the control mice developed autochthonous tumors. The tMK-2 cells also exerted in vitro NK-like cytotoxic activity, and their killing activity was strongly increased in the presence of both IL-2 and IL-12. These results suggest that the injected T-cells with TCR alpha/beta positive and CD4- /8- DN phenotype and NK-like activity are important in the therapy as well as the prevention of tumor development.

Functional and phenotypical characteristics of hepatic NK-like T cells in NK1.1-positive and -negative mouse strains.

We previously reported and partially characterized a unique monoclonal antibody (mAb), U5A2-13, which recognizes a T cell subset similar to NK1.1+ T cells, not only in NK1.1-positive mouse strains but also in NK1.1-negative strains. In NK1.1-positive C57BL/6 mice, U5A2-13+ TCRalphabeta+ cells produced abundant IL-4 as well as extremely high levels of IFN-gamma upon CD3 cross-linking, but this did not occur with U5A2-13- TCRalphabeta+ cells. In NK1.1-negative C3H/He mice, U5A2-13+ TCRalphabeta+ cells produced high levels of IL-4 and IFN-gamma upon CD3 cross-linking, but this was not observed with U5A2-13- TCRalphabeta+ cells. To the best of our knowledge, this is the first direct evidence of the presence of NK-like T cells defined phenotypically by U5A2-13 mAb and functionally by IL-4/IFN-gamma production in NK1.1-negative mouse strains. We also demonstrated that U5A2-13- NK1.1+ T cells and U5A2-13+ NK1.1- T cells in C57BL/6 mice could produce both IL-4 and IFN-gamma. In addition, Vbeta8 or Vbeta7 usage by U5A2-13+ NK1.1- T cells was lower than that by U5A2-13+ NK1.1+ T cells, but remained higher than that by U5A2-13- NK1.1- T cells. Based on the present results, U5A2-13 mAb appears to be a valuable tool in the study of NK-like T cells.

Mouse NK1.1+ cytotoxic T cells can be generated by IL-2 exposure from lymphocytes which express an intermediate level of T cell receptor.

NK-like T cells which express the NK1.1 molecule and CD3 (or TCR) of intermediate level (CD3int or TCRint cells) were recently demonstrated to be present in various immune organs, and to have NK-like cytotoxic activity against NK target cells. In this study, we investigated whether NK1.1- T cells could express NK1.1. We found that NK1.1+ TCRint cells were much more abundant in the liver (20%) than in the spleen (2%). When hepatic and splenic mononuclear cells (MNCs) were cultured either in the absence of IL-2 or in the presence of CD3/TCR cross-linking, the original NK1.1+ TCRint cells disappeared. However, when they were cultured in the presence of a high dose of IL-2 for 4 days, a new type of NK1.1+ T cell was formed to the extent of approximately 15-20%, and the liver and spleen contained similar percentages of this new type of NK1.1+ T cells. The phenotypes of the original and the new type of NK1.1+ T cells were clearly distinct. The freshly obtained NK1.1+ TCRint cells consisted of double-negative (DN) CD4-CD8- cells and single-positive (SP) CD4+ cells, whereas the new type of NK1.1+ T cells predominantly consisted of DN CD4-CD8- cells and SP CD8+ cells and expressed a high level of CD3 (CD3high or TCRhigh cells). When NK1.1- cells or IL-2 receptor beta-chain (IL-2Rbeta)- cells were isolated from the liver and spleen, and cultured in the presence of IL-2 for 4 days, NK1.1+ T cells were generated from NK1.1- cells, but not from IL-2Rbeta- cells. Our results suggested that the NK1.1- cells, but not IL-2Rbeta- cells, contained the precursor of IL-2-stimulated NK1.1+ TCRhigh cells. When purified NK1.1- IL-2Rbeta+ TCRint cells were cultured in the presence of IL-2 for 4 days, approximately 10% of the cells became NK1.1+ TCRhigh cells. Approximately 60% of the purified NK1.1+ TCRint cells lost NK1.1 expression. The IL-2-stimulated NK1.1+ TCRhigh cells that had arisen from NK1.1- TCRint cells exerted an NK cell-like cytotoxic activity similar to that of the original NK1.1+ T cells. Thus, NK1.1- TCRint cells could express NK1.1 and exert NK-like cytotoxic activity regardless of their origin. It appears that NK1.1+ TCRhigh cells can only be induced through an IL-2-stimulation pathway but not via CD3/TCR cross-linking.

A novel monoclonal antibody permitting recognition of NKT cells in various mouse strains.

By immunizing mouse lymphoma cell line tMK-2U derived from intermediate TCR cells of BALB/c nude mouse, U5A2-13 monoclonal antibody (mAb, a rat IgG2a) was established. U5A2-13 antigen (Ag) was expressed on around 65% of TCRint cells in the liver of the various mouse strains including both NK1.1- and NK1.1+ mouse strains, while NK1.1 Ag was expressed only in NK1.1+ C57BL/6 mouse strain. Among CD3+ cells, 26.3% cells co-expressed U5A2-13 Ag and NK1.1+ Ag, while small proportions of the CD3+ cells were U5A2-13+NK1.1- (9.2%) or U5A2-13-NK1.1+ (4.4%). Among NK1.1+ cells, 54.9% cells co-expressed CD3 and U5A2-13 Ag, while some proportions of the cells were U5A2-13+CD3- (19.4%) or U5A2-13-CD3+ (9.8%). It was found that approximately 85% of NK1.1+CD3+ cells coexpressed U5A2-13 Ag. U5A2-13 Ag with low fluorescence intensity was also expressed on 55% of NK1.1+CD3-NK cells. U5A2-13 Ag immunoprecipitated from tMK-2U cells consisted of three proteins, which were 65 kDa, 33 kDa and 32 kDa under both reducing and non-reducing conditions and these were apparently different from NK 1.1 Ag. These results indicated that U5A2-13 mAb was able to define a similar population to NK1.1+CD3+T cells and to 55% of NK1.1+CD3-NK cells in various strains, through recognizing a different molecule from NK1.1 Ag.

Amelioration of hemiplegia-associated osteopenia more than 4 years after stroke by 1 alpha-hydroxyvitamin D3 and calcium supplementation.

BACKGROUND AND PURPOSE: It has been demonstrated that bone mass was significantly reduced on the hemiplegic side of stroke patients, which might increase their risk of hip fracture. We evaluated the efficacy of 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3] and supplemental elemental calcium in maintaining bone mass and decreasing the incidence of hip fractures after hemiplegic stroke. METHODS: In a randomized study, 64 patients with hemiplegia after stroke with a mean duration of illness of 4.8 years received either 1 microgram 1 alpha (OH)D3 daily (treatment group, n = 30) or an inactive placebo (placebo group, n = 34) for 6 months and were observed for this duration. Both groups received 300 mg of elemental calcium daily. The bone mineral density (BMD) and metacarpal index (MCI) in the second metacarpals were determined by computed x-ray densitometry. The incidence of hip fractures in these patients was recorded. RESULTS: BMD on the hemiplegic side decreased by 2.4% in the treatment group and 8.9% in the placebo group (P = .0021), while BMD on the intact side increased by 3.5% and decreased by 6.3% in the treated and placebo groups, respectively (P = .0177). In the treatment group, the difference in BMD between hemiplegic and nonhemiplegic sides decreased significantly compared with that before randomization. This difference increased in the placebo group. We observed a similar improvement in MCI in the treatment group but not in the placebo group. Four patients in the placebo group suffered a hip fracture compared with none in the treatment group (P = .0362). CONCLUSIONS: Treatment with 1 alpha (OH)D3 and supplemental elemental calcium can reduce the risk of hip fractures and can prevent further decreases in BMD and MCI on the hemiplegic side of patients with a long-standing stroke. Treatment also may improve these indices on the intact side.

Recurrence of spontaneous intracranial hypotension with subdural hematomas.

A 59-year-old man developed postural headache associated with a low CSF pressure. A CT scan revealed no abnormal findings and the orthostatic symptoms resolved without treatment 6 weeks after onset. He was diagnosed as having spontaneous intracranial hypotension (SIH) and remained symptom-free until he experienced recurrence of postural headache 9 months later. A lumbar puncture demonstrated low CSF pressure, and a CT scan revealed slit-like ventricles with narrowing of the sulci, Sylvian fissures, and infratentorial cisterns, in addition to bilateral subdural masses. After draining the hematomas, his symptoms resolved completely, and a follow-up CT scan was normal. We hypothesize that recurrent SIH in this case was due to small recurrent tears of a root sleeve. This case emphasizes the importance of follow-up of SIH for at least 9 months after resolution of symptoms.

[Clinical and pathological study of esophageal mucosa with hypopharyngeal cancer].

Hypopharyngeal cancer has been reported to be frequently associated with cancer of the upper gastrointestinal tract, especially the esophagus. We recently reviewed the records of patients who had undergone closed-chest esophagectomy to assess the value of endoscopy with iodine staining as a means of preoperative diagnosis of double cancers in this area and to investigate the characteristics of the hypopharyngeal and esophageal mucosa as sites for multicentric carcinogenesis. The subjects of this study were 30 patients who had undergone closed chest esophagectomy between January 1992 and December 1995 because of hypopharyngeal cancer. The following results were obtained: 1. Preoperative iodine staining often revealed the presence of cancer, with unstained areas covering more than half the circumference of the esophagus and being more than 3 cm in size. 2. Esophageal cancer and hypopharyngeal cancer were detected concurrently in 15 cases (synchronous double cancer) and at different times in 6 cases (metachronous double cancer). Synchronous esophageal cancer was more common in cases of advanced hypopharyngeal cancer, especially Stage IV. 3. When the number of cancer foci, their distribution along the circumference of the esophagus, and the extent of tumor spread in the esophagus were investigated, multiple and localized foci smaller than 1 cm were found to be more common in synchronous cancer, and solitary foci were more common in metachronous cancer. 4. The second primary esophageal cancer often occupied the Im or Ei area, and in metachronous double cancer, it was often localized in the middle and/or inferior segment of the esophagus. 5. In 60% of cases of synchronous double cancer, the esophageal cancer was confined to the mucosa. The esophageal cancer was early stage in 86.7% of cases of synchronous double cancer. These findings allow us to draw the following conclusions: (i) Because esophageal cancer which occurs synchronously with hypopharyngeal cancer tends to recur, it is suggested that a technique that allows complete extraction of the esophagus be selected. (ii) Local treatment such as endoscopic mucosal resection should be selected in metachronous early double cancer. (iii) Unstained areas extending along more than half the circumference of the esophagus and more than 3 cm in size suggest a high probability of the presence of cancer in this area. Adequate examination is needed in such cases. (iv) Screening of the upper gastrointestinal tract is important to detect head and neck cancer. An adequate examination schedule, tailored to the features of individual cases, seems essential.

Vitamin D deficiency and osteopenia in the hemiplegic limbs of stroke patients.

BACKGROUND AND PURPOSE: Little is known about bone changes in hemiplegic stroke patients. We evaluated the vitamin D status and bone changes on the hemiplegic and intact sides of stroke patients. METHODS: Sera were collected from 87 hemiplegic stroke patients (42 outpatients and 45 inpatients) and from 28 control subjects. The sera were assayed for 25-hydroxyvitamin D (25-OHD). Bone density was measured bilaterally from radiographs of the hands. Diet and sunlight exposure were assessed for all subjects. RESULTS: Serum 25-OHD concentrations were lower in patients (9.1 +/- 4.9 ng/mL for outpatients, 5.9 +/- 4.1 ng/mL for inpatients) than in control subjects (21.6 +/- 3.1 ng/mL). The difference in serum 25-OHD between the two patient groups also was statistically significant. The patients' microdensitometric scores for osteopenia were higher on the hemiplegic side than on the non-hemiplegic side. The microdensitometric scores and their side-to-side differences in patients correlated negatively with the serum 25-OHD concentration and positively with the degree of paralysis. Dietary intake of vitamin D was below the recommended level in 72% of the patients, and 89% of the patients were considered sunlight-deprived. CONCLUSIONS: Bone mass was reduced significantly on the hemiplegic side in the stroke patients, which might increase the risk of hip fracture. Vitamin D deficiency and disuse are the probable causes of osteopenia in this population. The hypovitaminosis D might be corrected readily by routine use of vitamin D supplements.

Development of osteopenia in the hemiplegic finger in patients with stroke.

Previous studies have demonstrated that hip fractures in stroke patients occurred almost exclusively on the hemiplegic side. We examined the bone changes in the second metacarpal of the hemiplegic side in terms of microdensitometric indices in 93 stroke patients with hemiplegia. All six indices indicated a significant decrease in bone mass on the hemiplegic side compared with the contralateral side. Differences in the indices between the hemiplegic and contralateral sides were correlated well with the duration of the illness and Brunstrom's stage for finger, arm, and leg. The same degree of osteopenia occurred in both small capsular and large hemispheric lesions. The same osteopenia was demonstrated in paralyzed and immobilized patients with myopathy. Thus a combination of weakness and immobilization is thought to be responsible for the osteopenia in the hemiplegic metacarpal bone. The osteopenia noted in the second metacarpal in the affected limb may account for the fact that hip fractures in stroke patients occur almost exclusively on the hemiplegic side.

Photodynamic therapy for head and neck cancer.

Photodynamic therapy (PDT) is a recently developed treatment involving the use of a photosensitizer and low power light, usually from a laser, to selectively destroy tumor cells. At present, we perform PDT for head and neck cancer using argon or excimer dye lasers with hematoporphyrin derivative as a photosensitizer. This study attempted to assess the utility and safety of PDT and to investigate the long-term outcome. All 24 patients had squamous cell carcinoma: 15 with laryngeal, 5 with lingual or oral, and 4 with pharyngeal cancer and were treated by PDT. Data were obtained from records from February 1988 through April 1995. After PDT, 12 of 15 laryngeal cancer patients were classified as having a complete remission (CR), as were 2 of the 5 lingual or oral and one of the 4 pharyngeal cancer patients. The patients were followed for 8 to 153 months. The longest duration of CR in patients treated by PDT alone was 148 months. Photosensitivity was experienced by all patients, but required no treatment. Liver, kidneys, and bone marrow showed no abnormal values. There were no clinically relevant adverse reactions, and patients with severe complications due to other types of treatment and elderly patients were also treated safely with this therapy.