Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair.

BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

Evaluation of the effects of glucose and oxygen on the vascular endothelial cell migration.

Glucose is essential as the main energy source for living organisms. However, excessive elevation of blood sugar levels can lead to diabetes and serious complications such as arteriosclerosis. Even though blood sugar levels as well as hypoxia associated with hyperglycemia are known to be closely related to diabetes complications, the responses of vascular endothelial cells to glucose and oxygen have not been fully investigated. In this study, using a microfluidic device that can control the oxygen concentration, we observed the behavior of vascular endothelial cell monolayers while simultaneously controlling glucose and oxygen levels. Results showed that the cell migration speed was increased by high-glucose exposure in an oxygen-rich environment, but was decreased in a hypoxic environment regardless of glucose condition. The expression of vascular endothelial-cadherin at the cell periphery, which plays a role in cell-cell adhesion, was increased by hypoxic exposure, but was largely independent of glucose condition. This suggested that cell-cell adhesion is less involved in the increase in migration caused by high glucose. Furthermore, stabilization and nuclear translocation of hypoxia-inducible factor-1α, which is involved in cellular hypoxia sensing, increased 5 h after exposure to high glucose, but decreased 3 days after the exposure. This indicated that intracellular hypoxia was generated by increased oxygen consumption in mitochondria just after the high-glucose exposure, but it was moderated within 3 days.

Multifunctional transcription factor TFII-I is an activator of BRCA1 function.

BACKGROUND: The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams-Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date. METHODS: A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated. RESULTS: We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Identification of DBC1 as a transcriptional repressor for BRCA1.

BACKGROUND: DBC1/KIAA1967 (deleted in breast cancer 1) is a putative tumour-suppressor gene cloned from a heterozygously deleted region in breast cancer specimens. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. Hereditary breast and ovarian cancer susceptibility gene product BRCA1, by binding to the promoter region of SIRT1, is a positive regulator of SIRT1 expression. METHODS: A physical interaction between DBC1 and BRCA1 was investigated both in vivo and in vitro. To determine the pathophysiological significance of DBC1, its role as a transcriptional factor was studied. RESULTS: We found a physical interaction between the amino terminus of DBC1 and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous DBC1 and BRCA1 form a complex in the nucleus of intact cells, which is exported to the cytoplasm during ultraviolet-induced apoptosis. We also showed that the expression of DBC1 represses the transcriptional activation function of BRCT by a transient expression assay. The expression of DBC1 also inhibits the transactivation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed that DBC1 may modulate the cellular functions of BRCA1 and have important implications in the understanding of carcinogenesis in breast tissue.

The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

A method using fibrin-fixed Blue Dextran for determining the plasmin and plasmin inhibitor activities in human plasma.

The fibrinolytic activity of plasmin was determined by incubating with fibrin-fixed Blue Dextran as a substrate, the Blue Dextran released being proportional to the plasmin activity. The applicability of this method for rapid and accurate evaluation of fibrinolytic activity was demonstrated by dose-response curves with purified plasmin, plasmin generated by urokinase in human plasma and euglobulin. The method can also be used to determined plasmin inhibitors in plasma.

Paradoxical response of growth hormone to peptide histidine methionine in acromegaly: comparison with the effects of thyrotropin-releasing hormone and vasoactive intestinal peptide.

We examined whether peptide histidine methionine (PHM) induces a paradoxical rise in plasma GH in patients with acromegaly. PHM (100 micrograms) was given as an iv bolus to eight patients with active acromegaly, and plasma GH levels were measured before and at intervals up to 120 min after the injection. For comparison, the effects of TRH (500 micrograms) and vasoactive intestinal peptide (VIP, 100 micrograms), peptides known to paradoxically stimulate GH secretion in acromegalics, were assessed in all of the patients. A paradoxical rise (greater than 50% above the basal) in plasma GH was observed in five patients after both TRH and VIP administrations, although TRH responders were not always VIP responders, nor did VIP responders always respond to TRH. In two patients, the GH response to PHM fulfilled the criteria of a paradoxical increase. Both of these patients were also TRH and VIP responders. These results suggest that PHM may be another hypothalamic hormone capable of paradoxically stimulating GH secretion in at least some acromegalics, although PHM appears to be a less potent stimulator of GH release than TRH and VIP. The pathophysiological significance of this phenomenon is yet to be determined.

Assessment of left ventricular function in Kawasaki disease by dipyridamole-loading cineventriculography.

Dipyridamole-loading cineventriculography was performed to evaluate left ventricular (LV) function in 76 patients with Kawasaki disease. Forty-five of 76 patients underwent the procedure within 6 months from the onset of illness (group 1A, normal findings on coronary angiogram; group 1B, maximal diameter of coronary lesions less than 4 mm; group 1C, maximal diameter of coronary lesions greater than 4 mm). Thirty-one of 76 patients underwent the procedure greater than 6 months after onset (group 2A, coronary lesions improved to normal or less than 4 mm; group 2B, remaining coronary lesions greater than 4 mm; group 2C, stenosis or obstruction of the coronary artery). Global LV ejection fraction increased in groups 1A, 1B and 2A after infusion of dipyridamole, but there was no significant change in groups 1C, 2B and 2C. In analyzing LV excursion, the pattern of LV wall motion with dipyridamole in groups 1B and 2A were similar to that in group 1A, but in 38% (5 of 13) of group 2A patients, abnormal regional LV wall motion after administration of dipyridamole was detected. Abnormal LV wall motion after dipyridamole infusion was detected in 42% (5 of 12) of group 1C patients, 38% (3 of 8 cases) of group 2B patients and 70% (7 of 10 cases) of group 2C patients. In conclusion, analysis of LV wall motion with dipyridamole is useful in evaluating cardiac involvement in Kawasaki disease.

Morphologic and functional assessment of coronary aneurysm after Kawasaki disease by repeated dipyridamole-loading coronary angiography.

The long-term outcome of coronary aneurysm after Kawasaki disease was investigated using dipyridamole-loading angiography in 33 children with coronary aneurysms after Kawasaki disease. Morphologic regression may not accompany functional improvement in the dilated coronary arteries.

Auditory brainstem responses in congenital heart disease.

To evaluate the effect of chronic hypoxemia on brainstem maturation, auditory brainstem responses were examined in 70 children (32 with and 38 without cyanosis) who had congenital heart disease. Ninety-one age-matched normal children served as controls. At 1-3 months of age, the I-V interpeak latencies of cyanotic infants (mean +/- S.D.; 5.17 +/- 0.17 ms) were more prolonged than were those of controls (4.95 +/- 0.11 ms) and those without cyanosis (4.84 +/- 0.22 ms; P < .05; P < .01). At 4-11 months of age, the I-V interpeak latencies of cyanotic infants (4.85 +/- 0.13 ms) were more prolonged than were those of controls (4.67 +/- 0.19 ms) and those not experiencing cyanosis (4.5 +/- 0.17 ms; P < .05; P < .01). In the cyanotic children, there was a significant negative correlation between the I-V interpeak latency and oxygen partial pressure (P < .01) or oxygen saturation (P < .05). Three of the 70 patients (4.3%) with congenital heart disease had absent auditory brainstem response. These data indicate that chronic hypoxemia may be one of the factors in retarded brainstem maturation.

Benefits of silicone cream occlusive dressing for treatment of meshed skin grafts.

A side-by-side evaluation of a silicone cream occlusive dressing technique for grafted meshed skin on the extremities of 10 patients is described. The site treated by silicone cream showed less congestion in the interstices of the mesh, less visible diamond pattern, and less hardness of the skin in all patients compared with the control site, which was treated by application of petroleum jelly ointment. The general appearance of the silicone cream-treated site showed better cosmetic results when compared with the control site.

Treatment of dermal depth burn wounds with an antimicrobial agent-releasing silicone gel sheet.

Silicone gel sheets containing 0.02 per cent Ofloxacin were used in the treatment of 24 patients with a total of 27 dermal depth burn wounds. The gel provided a continuing drug delivery system from the dressing to the wound. Clinically the silicone gel sheets did not adhere to the wound and could be removed easily without pain. No infection developed in any of the dermal depth burn wounds treated with the gel sheets. The silicone gel sheets were found to promote prompt epithelialization in 16 burn wounds of superficial dermal depth (mean 8.4 days) compared with ointment-impregnated gauze dressings (mean 14 days, P less than 0.01). There was less pain and discharge by macroscopic observation of the absorbent materials from both dressings. In nine wounds of mixed superficial and deep dermal burn, the silicone gel also provided prompt epithelialization (mean 12 days) compared to the control wounds (mean 22 days, P less than 0.01).

Experiences using silicone gel tie-over dressings following skin grafting.

Twenty-six patients with 27 skin grafts treated with tie-over dressings using silicone gel sheets containing 0.02 per cent Ofloxacin are described. This method ensured direct in-progress inspection of healing of the grafted skin. Although the silicone gel tie-over procedure without any additional pressure resulted in haematoma and congestion in four out of 12 grafts (33.3 per cent), in the group with additional pressure (approximately 20-30 mmHg) limited to the early period after surgery there was no haematoma and congestion of the grafts in 15 grafts (P less than 0.05, Chi-square test). It is surmised that adequate pressure applied to the graft for a few days after surgery would be beneficial in preventing haematoma formation.

Role of rBAT gene products in cystinuria.

To investigate whether rBAT gene products function as a crystine transporter component or as a transport activator, we microinjected several C-terminal deletion mutants of rBAT cRNA into Xenopus oocytes, and measured transport activity for arginine, leucine and cystine in the presence and absence of sodium. Wild type rBAT significantly stimulated the uptake of all 3 amino acids 10-20 fold compared to control mutants. On the other hand, no mutant, except a Δ511-685 mutant, stimulated the uptake of these amino acids. However, the Δ511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Δ511-685 mutant also increased the uptake of leucine. The Δ511-685 mutant did not stimulate crystine uptake in the presence and absence of sodium. Furthermore, inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium. These results suggest that mutant rBAT stimulates the endogenous amino acid transport system y+ in oocytes. Finally, rBAT gene products, as the primary cause of cystinuria, may function as activators of the amino acid transport system in renal brush border membrane.

Measurement of beta 2-microglobulin in bovine serum and urine by radioimmunoassay.

A radioimmunoassay (RIA) system for quantification of bovine beta 2-microglobulin (beta 2-M) in serum and urine was developed. The protein isolated from bovine colostrum showed a single band in SDS-PAGE, and its molecular weight was approximately 11,600. Amino acid sequences for the first 24 residues and the amino acid composition of the protein were in agreement with those in the bovine beta 2-M of previous research works. In an Ouchterlony test, a single precipitation line was formed between the protein and the antiserum made by the protein. From these results, it was confirmed that the protein isolated from the colostrum was pure bovine beta 2-M. For creation of an RIA calibration curve for urine, a urine void of beta 2-M, as much as possible (beta 2-M-free urine), and a PBS were used as diluents. Intraassay (n = 10) and interassay (n = 3) variances were 1.7-4.6% and 7.1-11.5% in the PBS dilute method, and were 1.4-5.1% and 12.3-13.5% in the beta 2-M-free urine dilute method, respectively. Mean recoveries were 160 +/- 19% (mean +/- SD) and 98.4 +/- 7.9% in PBS and beta-M-free urine, respectively. It was found that the method using the beta 2-M-free urine as a diluent was more accurate than using PBS. The beta 2-M concentrations in serum and urine of healthy Holstein cows measured by this RIA system showed a logarithmic normal distribution for urine and a normal distribution for serum. The mean beta 2-M concentrations were 0.0305(+0.04443)(-0.0210) mg/l (Geometric mean +/- S.D., n = 43) in urine and 2.87 +/- 0.45 mg/l (Arithmetic mean +/- S.D., n = 26) in sera. Further, we could not observe the particular tendency of daily variation in urinary beta 2-M concentrations of healthy cows (Holstein, n = 3 x 2 days).

Psychological evaluation of Japanese astronaut applicants.

In 1991, a new psychological selection procedure was employed during the selection of Japanese Space Station astronauts. It was based on international selection criteria developed by an international psychological/psychiatric working group. A total of 372 individuals--fewer than expected--submitted applications for Space Station astronaut in Japan. Of the applicants, 233 were given several psychological written tests [Anxiety Scale, Performance Test, General Aptitude Test Battery, and Environmental Adjustment Test Battery (EATB)] in Phase I of the selection. Forty-five applicants went on to take the General Aptitude Test Battery (GATB), Human Assessment Method (a group test), a semi-structured psychological interview and an intelligence test in Phase II of the selection. All applicants were found to be highly intelligent. Interestingly, an unexpectedly large number of candidates were disqualified by the newly developed EATB. Assessment of individual functioning in a group (the Human Assessment Method) resulted in no applicant being ranked in either the "qualified with reservation" (QR) or "disqualified" (DQ) category. Much has been learned from this initial application of psychological "select-in" testing, but further efforts are needed to improve both psychological criteria and evaluation methods and to determine their reliability and validity.

[Ventricular septal defect associated with pulmonary hypertension in monozygotic twins--a case report with surgical repair].

This is a report of two-year-old female monozygotic twins, both having a ventricular septal defect (VSD, Kirklin type II) and accompanying moderate pulmonary hypertension (PH). The two patients underwent a patch closure procedure to repair the VSD at the same time, and their postoperative courses were uneventful. Rubenstein and Weaver have reported the only case of surgical treatment for monozygotic twins having both VSD and PH. Our patients are the second such case of surgical treatment of VSD and PH in monozygotic twins.

[Prefectural health administration's view on otorhinolaryngological care in medical rural areas].

In a previous study, we investigated otorhinolaryngological care by the general practitioners in medical rural areas and demonstrated that a close relationship between general practitioners and otorhinolaryngologists was necessary. In the present study, a survey of prefectural health administration's views on otorhinolaryngological care in medical rural areas was performed. A questionnaire for otorhinolaryngological care in medical rural areas was sent to the divisions of health administration in the 47 prefectures of Japan. Of these prefectures, 46 (97.9%) responded. About 80% of respondents have medical rural areas, and the otorhinolaryngological care is inadequate in most areas. General practitioners of internal medicine or surgery are primarily demanded in about 75% of the prefectures. Otorhinolaryngological care in medical rural areas has never been considered in most prefectures. Because the need for otorhinolaryngological medical care will increase, prefectural health administrations need to pay more attention to otorhinolaryngological care in medical rural areas, and a close relationship between general practitioners, otorhinolaryngologists, and the administration should be established.

Salivary gland gene expression atlas identifies a new regulator of branching morphogenesis.

During organ development, local changes in gene expression govern morphogenesis and cell fate. We have generated a microanatomical atlas of epithelial gene expression of embryonic salivary glands. The mouse submandibular salivary gland first appears as a single mass of epithelial cells surrounded by mesenchyme, and it undergoes rapid branching morphogenesis to form a complex secretory organ with acini connected to an extensive ductal system. Using laser capture microdissection, we collected samples from 14 distinct epithelial locations at embryonic days 12.5, 13.5, 14, and 15, and characterized their gene expression by microarray analysis. These microarray results were evaluated by qPCR of biological replicates and by comparisons of the gene expression dataset with published expression data. Using this gene expression atlas to search for novel regulators of branching morphogenesis, we found a substantial reduction in mRNA levels of GSK3ß at the base of forming clefts. This unexpected finding was confirmed by immunostaining, and inhibition of GSK3ß activity enhanced salivary gland branching. This first microanatomical expression atlas of a developing gland characterizes changes in local gene expression during salivary gland development and differentiation, which should facilitate the identification of key genes involved in tissue morphogenesis.