Pretreatment with buthionine sulfoximine enhanced uptake and retention of BSH in brain tumor.

To determine the influence of buthionine sulfoximine (BSO) on boron biodistribution after sulfhydryl borane (BSH) administration for boron neutron capture therapy, the effectiveness of the combination of BSO with sulfhydril- (BSH) and non-sulfhydril (B12H12 and BNH3) boron compounds, and the interval between BSO and BSH administration, the retention of boron in tissues have been evaluated using a 9L rat tumor model. Simultaneous administration of BSH and BSO showed significantly higher boron accumulation compared to that without BSO, however there was no difference in tissue boron level between B12H12 and BNH3 administration with BSO or without BSO. The longer interval (6h) between BSH and BSO administration related to the highest boron concentration in the brain and subcutaneous tumors compared to shorter intervals (0.5, 3h). Boron concentration in subcutaneous and brain tumors was maintained for 6 and 12h after the administration of BSH following BSO pretreatment.

Acute type-A aortic dissection with patent false lumen through to the abdominal aorta: effects of a conventional elephant trunk on malperfusion syndromes and narrowed true lumen.

BACKGROUND: Narrowed true lumen and patent false lumen through to the terminal aorta is a high-risk condition for malperfusion syndromes (MS) in acute type-A aortic dissection. It is important to ascertain how the true and false lumens behave after surgery. PATIENTS AND METHODS: We retrospectively investigated 45 patients with this pathology. The true lumen sizes at the narrowest levels above and below the superior mesenteric artery were followed by computed tomography after surgery (0-36 months). RESULTS: Thirty-seven MS were seen in 23 patients. Hospital mortality was 8.9%. The narrowed true lumen was not enlarged in the first 6 months with a patent false lumen. The elephant trunk procedure did not improve the true lumen size. An extremely narrowed (≤3 mm) true lumen was associated with a significantly high incidence of MS and mortality. CONCLUSIONS: High incidences of MS were observed in this particular pathology. An extremely narrowed true lumen was accompanied by a high incidence of MS and mortality.

Cytokeratins 18 and 8 are poor prognostic markers in patients with squamous cell carcinoma of the oesophagus.

BACKGROUND: Cytokeratins (CKs) are structural marker proteins specific for epithelial cells. However, recent studies indicate their involvement in cancer progression. METHODS: We evaluated CK18 and its filament partner, CK8 expression, by immunohistochemistry in 210 resected specimens from patients with oesophageal squamous cell carcinoma (OSCC). We also analysed the relationship between their expression and various clinicopathological parameters including prognosis. RESULTS: Neither CK18 nor CK8 was expressed in non-cancerous squamous epithelium whereas proper oesophageal glands expressed both CKs. Ninety (42.9%) tumours were CK18 positive and 85 (40.5%) CK8 positive, and the concordance rate for immunohistochemical classification for CK18 and CK8 was 82.4%. CK18 expression correlated with poorly differentiated tumours, use of neo-adjuvant chemotherapy, and advanced stage. Prognosis of patients with CK18-positive tumours was poorer than that of patients with negative OSCC (P<0.001). A similar trend was noted for CK8 expression. Multivariate analysis identified pT (P=0.020), pN number (P=0.001), and CK18 expression (P=0.004) as independent prognostic factors. CK18 expression in 83 pretreatment biopsy specimens was detected in 47 cases (56.6%) and also correlated with prognosis (P=0.045). CONCLUSION: CK18/CK8 expression correlated with progression of OSCC. The significant correlation with prognosis and stable expression in biopsy specimen suggest usefulness of CK18 in selection of treatment strategies for OSCC.

Intracellular uptake of a new boronated porphyrin EC032.

We measured the toxicity and intracellular uptake of a newly developed boronated porphyrin EC032, and verified the fluorescence-based boron concentration measuring methods. Toxicity study showed that concentration required to produce a 50% reduction in viability (IC(50)) of EC032 was more than 0.25 mM. Fluorescence study showed the intracellular uptake of EC032 increased up until 24 h after its exposure to C6, 9L, U87, and U251 cells. There was also a linear correlation between ICP-AES and fluorescence intensity as an arbitrary unit about measurement of boron concentration. Fluorescence-based boron concentration measuring methods are very simple and useful methods, especially for screening of slight test dose of porphyrin compounds.

Boron neutron capture therapy for newly diagnosed glioblastoma: a pilot study in Tsukuba.

Neutron capture therapy (NCT) theoretically allows an unique tumor-cell-selective high-LET particle radiotherapy. The survival benefits and safety of NCT were evaluated in 15 patients with newly diagnosed glioblastoma multiforme (GBM). Seven patients received intra-operative (IO-) NCT and eight patients received external beam (EB-) NCT. Sulfhydryl borane (BSH, 5 g/body) was administered intravenously 12 h before neutron irradiation. Additionally, p-dihydroxyboryl-phenylalanine (BPA, 250 mg/kg) was given 1 h before irradiation to the eight patients who underwent EB-NCT. EB-NCT was combined with fractionated photon irradiation. Five of 15 patients were alive at analysis for a mean follow-up time of 20.3 M. In 11 of 15 patients followed up for more than 1-year, eight (72.7%) maintained their Karnofsky performance status (KPS; 90 in 6 and 100 in 2). The median overall survival (OS) and time to magnetic resonance (MR) change (TTM) for all patients were 25.7 and 11.9 M, respectively. There was no difference in TTM between the IO-NCT (12.0 M) and EB-NCT (11.9 M) groups. The 1- and 2-year survival rates were 85.7% and 45.5%, respectively. This NCT pilot study in 15 patients with newly diagnosed GBM showed survival benefits, suggesting that the neutron capture reaction may function sufficiently to control tumors locally, and that further optimized studies in large series of patients are warranted.

Dose distribution and clinical response of glioblastoma treated with boron neutron capture therapy.

The dose distribution and failure pattern after treatment with the external beam boron neutron capture therapy (BNCT) protocol were retrospectively analyzed. BSH (5 g/body) and BPA (250 mg/kg) based BNCT was performed in eight patients with newly diagnosed glioblastoma. The gross tumor volume (GTV) and clinical target volume (CTV)-1 were defined as the residual gadolinium-enhancing volume. CTV-2 and CTV-3 were defined as GTV plus a margin of 2 and 3 cm, respectively. As additional photon irradiation, a total X-ray dose of 30 Gy was given to the T2 high intensity area on MRI. Five of the eight patients were alive at analysis for a mean follow-up time of 20.3 months. The post-operative median survival time of the eight patients was 27.9 months (95% CI=21.0-34.8). The minimum tumor dose of GTV, CTV-2, and CTV-3 averaged 29.8+/-9.9, 15.1+/-5.4, and 12.4+/-2.9 Gy, respectively. The minimum tumor non-boron dose of GTV, CTV-2, and CTV-3 averaged 2.0+/-0.5, 1.3+/-0.3, and 1.1+/-0.2 Gy, respectively. The maximum normal brain dose, skin dose, and average brain dose were 11.4+/-1.5, 9.6+/-1.4, and 3.1+/-0.4 Gy, respectively. The mean minimum dose at the failure site in cases of in-field recurrence (IR) and out-field recurrence (OR) was 26.3+/-16.7 and 14.9 GyEq, respectively. The calculated doses at the failure site were at least equal to the tumor control doses which were previously reported. We speculate that the failure pattern was related to an inadequate distribution of boron-10. Further improvement of the microdistribution of boron compounds is expected, and may improve the tumor control by BNCT.

Distance determination in human ubiquitin by pulsed double electron-electron resonance and double quantum coherence ESR methods.

Recently, distance measurements by pulsed ESR (electron spin resonance) have been obtained using pulsed DEER (double electron-electron resonance) and DQC (double quantum coherence) in SDSL (site directed spin labeling) proteins. These methods can observe long range dipole interactions (15-80A). We applied these methods to human ubiquitin proteins. The distance between the 20th and the 35th cysteine was estimated in doubly spin labeled human ubiquitin. Pulsed DEER requires two microwave sources. However, a phase cycle is not usually required in this method. On the other hand, DQC-ESR at X-band ( approximately 9GHz) can acquire a large echo signal by using pulses of short duration and high power, but this method has an ESEEM (electron spin echo envelope modulation) problem. We used a commercial pulsed ESR spectrometer and compared these two methods.

[Advantages of side-to-side anastomosis in the assessment and revision of coronary artery bypass grafting].

With the advent of drug eluting stents in percutaneous coronary intervention, required quality level of coronary artery bypass grafting (CABG) has been elevating. To obtain nearly perfect patency of bypass grafts, intraoperative assessment and repair of abnormal grafts are helpful. We report systematic revision and safe repair technique of arterial grafts in CABG. Side-to-side instead of commonly used end-to-side anastomosis of arterial grafts is the first step in this technique. When any abnormalities are noted in intraoperative flowmetry of a graft, the distal surgical clip is removed. Free flow of the graft is measured. A coronary probe is gently inserted into the graft and the coronary artery. Vasodilators can be injected into the graft if necessary. When direct revision of the anastomosis is indicated, the graft is cut longitudinally from the distal end up to just proximal to the anastomotic site. The shape of the anastomosis can be observed directly without removing sutures. When re-anastomosis is not indicated, the distal remnant graft tissue is folded back and utilized as a patch. Thus the graft can be easily closed without narrowing.

[The correlation between flow pattern during cardiopulmonary bypass and patency of the coronary artery bypass grafts].

Recently the availability of transit time flow measurement (TTFM) is reported especially in off-pump coronary artery bypass grafting (CABG). But little is known about TTFM findings in on-pump CABG. We examined the correlation between the TTFM flow pattern and the angiography findings in on-pump CABG. The subjects consisted of 52 patients who underwent on-pump CABG and angiography early after operation. In these patients, 55 internal thoracic artery (ITA), 17 gastroepiploic artery (GEA), 13 saphenous vein graft (SVG) and 41 radial artery (RA) were tested with TTFM during cardiopulmonary bypass (CPB). TTFM demonstrated a diastolic filling pattern in 53 ITA, 16 GEA, 13 SVG and 36 RA. The angiography revealed that all these grafts were perfectly patent with the exception of a GEA with a flow competition pattern. TTFM revealed an abnormal flow pattern in 2 ITA (these 2 grafts were revised during CPB and the angiography demonstrated their perfect patency), 1 GEA (to and fro pattern), 0 SVG and 5 RA (the abnormal pattern was due to graft spasm in 3 of 5, and the angiography revealed their perfect patency, however, the angiography detected stenosis in the remaining 2 grafts). The present study found that the TTFM flow pattern during CPB correlated well with the angiography findings. TTFM during CPB was useful to detect graft failure, and grafts were revised safely during CPB.

Activation of WNT family expression and signaling in squamous cell carcinomas of the oral cavity.

The WNT family activates an oncogenic signaling mediated through beta-catenin and is up-regulated in a variety of malignant neoplasms. The signaling translocates beta-catenin into the nucleus and stimulates carcinoma cells in the epithelial-mesenchymal transition (EMT). However, WNT expression and signaling in oral carcinomas have not been examined. The present study focused on unveiling the involvement of WNTs in oral carcinomas, and showed that carcinoma cells express 11 of 19 WNT family members by reverse-transcription/PCR. WNT-expressing carcinoma cells exhibited increased beta-catenin levels in the cytoplasmic pool and translocation to the nucleus. The activation state of signaling correlated with the expression of membrane-type 1 matrix metalloproteinase, which degrades territorial matrices in carcinoma invasion. Immunohistochemistry disclosed that WNT3 expression and nuclear localization of beta-catenin were predominant in carcinoma cells at the invasive front. These results suggest that enhanced WNT expression and signaling accelerate the progression of carcinomas via activating EMTs and local invasiveness.

Regulation of transcription and chromatin by methyl-CpG binding protein MBD1.

DNA methylation is important for epigenetic regulation of genome, and it is interpreted by specific protein factors that contain a highly conserved methyl-CpG binding domain (MBD). There are at present five mammalian MBD family proteins: MBD1, MBD2, MBD3, MBD4 and MeCP2. In the family of methyl-CpG binding proteins, MBD1 is characterized by the presence of MBD, two or three cysteine-rich CXXC motifs, and the C-terminal transcriptional repression domain (TRD). In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the existence of CXXC1, CXXC2, and CXXC3 in MBD1 isoform v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and MBD1v4. MBD1v1 represses transcription preferentially from both unmethylated and hypomethylated promoters, while MBD1v3 inhibits hypermethylated but not unmethylated promoter activities. The MBD and CXXC3 sequences are responsible for the ability to bind methylated and unmethylated DNAs, respectively. MBD1 is also found to be a chromosomal protein that forms many foci within the nucleus. These findings suggest that MBD1 is a unique transcriptional regulator depending on the density of methyl-CpG pairs.

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups.

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1--21)NH(2), bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3--7 form a unique beta-coil structure that is preceded by an N-terminal extended tail. This beta-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8--21) forms an alpha-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA.

Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.

Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1.

MBD1 is a mammalian protein that binds symmetrically methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MBD1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation-dependent gene repression. Based on these findings we have concluded that MBD1 acts as a transcriptional regulator depending on the density of methyl-CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.

Solution structure of the methyl-CpG-binding domain of the methylation-dependent transcriptional repressor MBD1.

CpG methylation in vertebrates is important for gene silencing, alterations in chromatin structure and genomic stability, and differences in the DNA-methylation status are correlated with imprinting phenomena, carcinogenesis and embryonic development. Methylation signals are interpreted by protein factors that contain shared methyl-CpG-binding domains (MBDs). We have determined the solution structure of the MBD of the human methylation-dependent transcriptional repressor MBD1 by multi-dimensional heteronuclear NMR spectroscopy. It folds into an alpha/beta-sandwich structure with characteristic loops. Basic residues conserved in the MBD family are largely confined to one face of this fold and a flexible loop, which together form a large positively charged surface. Site-directed mutagenesis and chemical shift changes upon complexing with a methylated DNA facilitated identification of this surface as the DNA interaction site. In addition to three basic residues, conserved Tyr34 and Asp32 were shown to be important for the DNA binding.

The role of human MBF1 as a transcriptional coactivator.

Multiprotein bridging factor 1 (MBF1) is a coactivator which mediates transcriptional activation by interconnecting the general transcription factor TATA element-binding protein and gene-specific activators such as the Drosophila nuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Here we report the cDNA cloning and functional analysis of hMBF1. Two isoforms, which we term hMBF1alpha and hMBF1beta, have been identified. hMBF1alpha mRNA was detected in a number of tissues, whereas hMBF1beta exhibited tissue-specific expression. Both isoforms bound to TBP and Ad4BP/SF-1, a mammalian counterpart of FTZ-F1, and mediated Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 was detected in the cytoplasm by immunostaining, coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induced accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus through its binding to Ad4BP/SF-1. hMBF1 also bound to ATF1, a member of the basic leucine zipper protein family, and mediated its activity as a transcriptional activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes.

Resonance assignments, solution structure, and backbone dynamics of the DNA- and RPA-binding domain of human repair factor XPA.

XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (Dparallel/Dperpendicular) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.

Solution structure of the DNA- and RPA-binding domain of the human repair factor XPA.

The solution structure of the central domain of the human nucleotide excision repair protein XPA, which binds to damaged DNA and replication protein A (RPA), was determined by nuclear magnetic resonance (NMR) spectroscopy. The central domain consists of a zinc-containing subdomain and a C-terminal subdomain. The zinc-containing subdomain has a compact globular structure and is distinct from the zinc-fingers found in transcription factors. The C-terminal subdomain folds into a novel alpha/beta structure with a positively charged superficial cleft. From the NMR spectra of the complexes, DNA and RPA binding surfaces are suggested.