TTF-1/NKX2-1 binds to DDB1 and confers replication stress resistance to lung adenocarcinomas.

TTF-1, also known as NKX2-1, is a transcription factor that has indispensable roles in both lung development and physiology. We and others have reported that TTF-1 frequently exhibits high expression with increased copy number in lung adenocarcinomas, and also has a role as a lineage-survival oncogene through transcriptional activation of crucial target genes including ROR1 and LMO3. In the present study, we employed a global proteomic search for proteins that interact with TTF-1 in order to provide a more comprehensive picture of this still enigmatic lineage-survival oncogene. Our results unexpectedly revealed a function independent of its transcriptional activity, as TTF-1 was found to interact with DDB1 and block its binding to CHK1, which in turn attenuated ubiquitylation and subsequent degradation of CHK1. Furthermore, TTF-1 overexpression conferred resistance to cellular conditions under DNA replication stress (RS) and prevented an increase in consequential DNA double-strand breaks, as reflected by attenuated induction of pCHK2 and γH2AX. Our findings suggest that the novel non-transcriptional function of TTF-1 identified in this study may contribute to lung adenocarcinoma development by conferring tolerance to DNA RS, which is known to be inherently elicited by activation of various oncogenes.

ß-Decay Half-Lives of 110 Neutron-Rich Nuclei across the N=82 Shell Gap: Implications for the Mechanism and Universality of the Astrophysical r Process.

The ß-decay half-lives of 110 neutron-rich isotopes of the elements from _{37}Rb to _{50}Sn were measured at the Radioactive Isotope Beam Factory. The 40 new half-lives follow robust systematics and highlight the persistence of shell effects. The new data have direct implications for r-process calculations and reinforce the notion that the second (A≈130) and the rare-earth-element (A≈160) abundance peaks may result from the freeze-out of an (n,γ)⇄(γ,n) equilibrium. In such an equilibrium, the new half-lives are important factors determining the abundance of rare-earth elements, and allow for a more reliable discussion of the r process universality. It is anticipated that universality may not extend to the elements Sn, Sb, I, and Cs, making the detection of these elements in metal-poor stars of the utmost importance to determine the exact conditions of individual r-process events.

Effect of age on bowel management in traumatic central cord syndrome.

STUDY DESIGN: A retrospective multicenter study. OBJECTIVES: To investigate the characteristics of bowel dysfunction in elderly people with traumatic central cord syndrome (TCCS). SETTING: A total of 28 Rosai hospitals in Japan. METHODS: The Rosai Hospital registry included 3006 persons with spinal cord injury during 1997-2007. The study subjects were 186 patients with TCCS (160 men, 26 women; mean age, 61.7±11.6 years, ±s.d.). Patients were divided according to age into the young group (<50 years, n=30), the middle-age group (50-69 years, n=112) and the elderly group (≥70 years, n=44). We assessed the differences in bowel management techniques (spontaneous, rectal medications and manual emptying) and activity of daily living (ADL) with respect to bowel care at discharge among the three groups. RESULTS: Continent spontaneous defecation was the most common bowel management method (50%, 93/186). The percentage of elderly subjects on continent spontaneous defecation (36.4%) was significantly less than that of the young group (66.7%; P<0.05). Furthermore, the percentage of elderly patients who required no bowel care (18.2%) was significantly less than those of the young (53.3%) and middle-age groups (41.1%; P<0.01). However, few differences in bowel care-related ADL were recognized among the three groups in patients who required manual emptying. CONCLUSION: The results identified significantly fewer patients aged ≥70 years with 'continent spontaneous defecation' or 'independent for bowel care' compared with younger patients. The results also highlighted the clinical importance of bowel dysfunction associated with TCCS especially in elderly people.

Supernovae and the chirality of the amino acids.

A mechanism for creating amino acid enantiomerism that always selects the same large-scale chirality is identified, and subsequent chemical replication and galactic mixing that would populate the Galaxy with the predominant species is described. This involves (1) the spin of the 14N in the amino acids, or in precursor molecules from which amino acids might be formed, that couples to the chirality of the molecules; (2) the neutrinos emitted from the supernova, together with the magnetic field from the nascent neutron star or black hole formed from the supernova, which selectively destroy one orientation of the 14N and thus select the chirality associated with the other 14N orientation; (3) chemical evolution, by which the molecules replicate and evolve to more complex forms of a single chirality on a relatively short timescale; and (4) galactic mixing on a longer timescale that mixes the selected molecules throughout the Galaxy.

TAK1 kinase determines TRAIL sensitivity by modulating reactive oxygen species and cIAP.

TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of cell death in several cancer cells, but many cells are resistant to TRAIL. The mechanism that determines sensitivity to TRAIL-killing is still elusive. Here we report that deletion of TAK1 kinase greatly increased activation of caspase-3 and cell death after TRAIL stimulation in keratinocytes, fibroblasts and cancer cells. Although TAK1 kinase is involved in NF-kappaB pathway, ablation of NF-kappaB did not alter sensitivity to TRAIL. We found that TRAIL could induce accumulation of reactive oxygen species (ROS) when TAK1 was deleted. Furthermore, we found that TAK1 deletion induced TRAIL-dependent downregulation of cIAP, which enhanced activation of caspase-3. These results show that TAK1 deletion facilitates TRAIL-induced cell death by activating caspase through ROS and downregulation of cIAP. Thus, inhibition of TAK1 can be an effective approach to increase TRAIL sensitivity.

Evidence for nucleosynthesis in the supernova gamma process: universal scaling for p nuclei.

Analyzing the solar system abundance, we find two universal scaling laws concerning the p and s nuclei. They indicate that the gamma process in supernova (SN) explosions is the most probable origin of the p nuclei that has been discussed with many possible nuclear reactions and sites in about 50 years. In addition, the scalings lead to new concepts: a universality of the gamma process and a new nuclear cosmochronometer. We carry out gamma-process nucleosynthesis calculations for typical core-collapse SN explosion models, and the results satisfy the observed scalings.

The spin temperature of NH3 in Comet C/1999S4 (LINEAR).

A high-dispersion spectrum of Comet C/1999S4 (LINEAR) was obtained in the optical region with the high-dispersion spectrograph on the Subaru telescope when the comet was 0.863 astronomical units from the Sun before its disintegration. We obtained high signal-to-noise ratio emission lines of the cometary NH2 bands from which an ortho-to-para ratio (OPR) of 3.33 +/- 0.07 was derived on the basis of a fluorescence excitation model. Assuming that cometary NH2 mainly originates from ammonia through photodissociation, the derived OPR of NH2 molecules should reflect that of ammonia, which provides information on the environment of molecular formation or condensation and of the thermal history of cometary ices. Assuming that the OPR of ammonia in comets was unchanged in the nucleus, the derived spin temperature of ammonia (28 +/- 2 kelvin) suggests that a formation region of the cometary ammonia ice was between the orbit of Saturn and that of Uranus in the solar nebula.

New pulp biobleaching system involving manganese peroxidase immobilized in a silica support with controlled pore sizes.

Attempts have been made to use manganese peroxidase (MnP) for chlorine-free pulp biobleaching, but they have not been commercially viable because of the enzyme's low stability. We developed a new pulp biobleaching method involving mesoporous material-immobilized manganese peroxidase from Phanerochaete chrysosporium. MnP immobilized in FSM-16, a folded-sheet mesoporous material whose pore size is nearly the same as the diameter of the enzyme, had the highest thermal stability and tolerance to H(2)O(2). MnP immobilized in FSM-16 retained more than 80% of its initial activity even after 10 days of continuous reaction. We constructed a thermally discontinuous two-stage reactor system, in which the enzyme (39 degrees C) and pulp-bleaching (70 degrees C) reactions were performed separately. When the treatment of pulp with MnP by means of the two-stage reactor system and alkaline extraction was repeated seven times, the brightness of the pulp increased to about 88% within 7 h after completion of the last treatment.

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

Efficient production of artificially designed gelatins with a Bacillus brevis system.

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Meson Synchrotron Emission from Central Engines of Gamma-Ray Bursts with Strong Magnetic Fields.

Gamma-ray bursts (GRBs) are presumed to be powered by the still unknown central engines with timescales in the range from 1 ms to approximately a few seconds. We propose that the GRB central engines would be a viable site for strong meson synchrotron emission if they were compact astrophysical objects, such as neutron stars or rotating black holes with extremely strong magnetic fields (H approximately 1012-1017 G), and if protons or heavy nuclei were accelerated to ultrarelativistic energies on the order of approximately 1012-1022 eV. We show that the charged scalar mesons like pi+/- and heavy vector mesons like rho, which have several decay modes onto pi+/-, could be emitted, with a high intensity that is a thousand times larger than photons, through strong couplings to ultrarelativistic nucleons. These meson synchrotron emission processes eventually produce a burst of very high energy cosmic neutrinos with 1012 eV

Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant.

The efficient production of a thermostable protein disulfide isomerase (PDI) was successfully achieved using the newly isolated protease-deficient mutant, Bacillus brevis 31-OK. Extracellular protease (exoprotease) activity was about a quarter of that in the parent, and the mutant was deficient in at least one of the major exoproteases. The cDNA encoding the fungal PDI was inserted downstream of the signal peptide-encoding region in an expression-secretion vector for B. brevis. Efficient production of PDI was feasible using B. brevis 31-OK as a host and modified signal sequences composed of three leucine residues inserted in the hydrophobic region of the MWP (middle wall protein) signal sequence. The maximal secretion of PDI into the culture medium was 1.1 g/l, which is about twice that by the parent strain and fifty times greater than the amount of rat and murine PDIs produced by Escherichia coli. The enzymatic properties such as the specific activity and thermal stability of the recombinant PDI are similar to those of natural PDI derived from Humicola insolens mycelia. B. brevis 31-OK was able to maintain its exoprotease activity at a low level throughout the cultivation and is considered to be useful host for production of a protease-sensitive protein and for increase of protein productivity due to stable accumulation.

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system.

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L-1 by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system.

[Combination therapy with irinotecan and CBDCA for patients in terminal stage of ovarian carcinoma].

Combination chemotherapy with irinotecan and CBDCA was performed for two patients in the terminal stage of ovarian carcinoma. Every week, 75 mg.m-2 of irinotecan and 75-100 mg.m-2 of CBD CA were intravenously administered. Both patients showed a partial response. Diarrhea was not a characteristic side effect of irinotecan in this case. Dose limiting factors were leucopenia and thrombocytopenia.

Molecular cloning of a fungal cDNA encoding protein disulfide isomerase.

Based on the partial amino acid sequences of a protein disulfide isomerase (PDI) from Humicola insolens, two primers were synthesized for reverse transcriptase mediated polymerase chain reaction (RT-PCR) of a fungal RNA. A 0.2-kbp fragment around the consensus sequence of PDIs was obtained and used as a probe for screening a fungal cDNA library. A cDNA clone of PDI from H. insolens was isolated and encoded a polypeptide consisting of 505 amino acids, which was characterized by a N-terminal signal sequence composed of 20 amino acids, a consensus sequence (WCGHCK) at two positions, and a C-terminal endoplasmic reticulum retention signal (HDEL). Bacillus brevis harboring an expression plasmid bearing the fungal PDI cDNA was prepared and its culture supernatant showed a significant PDI activity. This indicates that glycosylation of a fungal PDI is not essential for the enzymatic activity related to an interchange of disulfide bonds.

Purification of protein disulfide isomerase from a thermophilic fungus.

A protein disulfide isomerase (PDI) was purified to homogeneity from the thermophilic fungus Humicola insolens by a rapid three-step procedure, anion-exchange chromatography, concanavalin A-affinity chromatography, and reverse phase high performance liquid chromatography. Forty-one micrograms of PDI was obtained from 100 g of wet mycelium. Concanavalin A-Sepharose chromatography is available for purification of the fungal PDI, indicating that the enzyme is also glycosylated like the yeast PDI. The fungal PDI exists as a dimer (2 x 60 kDa), has a pI of 3.5, and is fairly heat-stable. The amino acid composition of the PDI is similar to those of yeast and bovine liver PDI, and the high content of acidic amino acid residues agrees with the lower acidic pI.

Polyclonal activation of IgM antibodies to phospholipids in patients with idiopathic fetal growth retardation.

Phospholipid fractions and antibodies to phospholipids in the sera of normal pregnant women and patients with intra-uterine growth retardation (IUGR) were examined. Significant elevation of serum phospholipids were observed in the second and third trimesters of normal pregnancy. Serum concentration of phosphatidylserine and sphingomyelin was significantly lower in the third trimester in patients with IUGR than in normal pregnant women. Remarkable concentrations IgG and IgA antibodies to phospholipids were not observed in the patients or in normal pregnant women. Polyclonal activation of IgM antibodies was detected in normal pregnant women in the first and second trimesters but less so in the third. The activation of IgM antibodies was not observed in patients with IUGR induced by severe preeclampsia, but significant levels of IgM antibodies were detected in the third trimester in patients with idiopathic IUGR. It is suggested that antibody formation to phospholipids in normal pregnant women is induced by elevation of phospholipids and is down-regulated in the third trimester. Failure to down-regulate IgM antibodies may be related to the pathogenesis of idiopathic IUGR.