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In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.

Competency to stand trial (CST) is a contentious pretrial issue, confounded by ambiguous legal standards and inaccurate clinical tools. This article systematically explores both the controlling case law and the evolving assessment instruments, mindful of where and how they advance (or not) effective courtroom decision making. In the wake of our analysis, we propose a series of (criminal justice) policy reforms that address the problems posed by the CST legal doctrine and assessment technology. In brief, we call for changes with the competency evaluation procedures, the psychological instruments, and the legal remedies and/or strategies made available to the accused. We conclude by arguing that these revisions must be implemented if meaningful reform is to be effected for defendants whose competency is called into question prior to a criminal trial.

Recently, 57 undergraduate students at the University of Michigan were assigned the task of solving a crystal structure, given only the electron density map of a 1.3 Å crystal structure from the electron density server, and the position of the N-terminal amino acid. To test their knowledge of amino acid chemistry, the students were not given the protein sequence. With minimal direction from the instructor on how the students should complete the assignment, the students fared remarkably well in this task, with over half the class able to reconstruct the original sequence with over 77% sequence identity, and with structures whose median ranked in the 91(st) percentile of all structures of comparable resolution in terms of structure quality. Fourteen percent of the students' structures produced Molprobity steric clash validation scores even better than that of the original structure, suggesting that multiple students achieved an improvement in the overall structure quality compared to the published structure. Students were able to delineate limiting case chemical environments, such as charged interactions or complete solvent exposure, but were less able to distinguish finer details of hydrogen bonding or hydrophobicity. Our results prompt several questions: why were students able to perform so well in their structural validation scores? How were some students able to outperform the 88% sequence identity mark that would constitute a perfect score, given the level of degenerate density or surface residues with poor density? And how can the methodology used by the best students inform the practices of professional X-ray crystallographers?

Competency to stand trial (CST) determinations require an adherence to legal adjudication standards and psychological assessments methods. However, the forensic decision making on these matters is fraught with complex and enduring dilemmas. Questions persist about the vague and ambiguous nature of the precedent-setting U.S. Supreme Court case law on mental illness and competency as applied to particular defendants. In addition, the clinical evaluation procedures pose sufficient validity and reliability problems that the assessment instruments themselves have been the source of considerable consternation. As a result, the medicolegal system, on occasion, has allowed controversial and profoundly disturbed mental health defendants to pro se their cases (e.g., Colin Ferguson) and/or has rendered questionable psychological screenings for high-profile CST cases (e.g., Theodore Kaczynski). In this article, the authors revisit the legal and psychological pitfalls attributable to the CST determination. They examine where and how the legal standard and the clinical evaluation procedures manufacture problematic results, particularly in well-publicized cases. They conclude by exploring the policy implications of their analysis for purposes of future criminal justice reform in this forensic mental health area.

Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA–mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.

Viktor M Pastukh1, Li Zhang2, Mykhaylo V Ruchko1, Olena Gorodnya1, Gina C Bardwell1, Rubin M Tuder2, Mark N Gillespie11Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL, USA; 2Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado at Denver, Aurora, CO, USAAbstract: Lung tissue from COPD patients displays oxidative DNA damage. The present study determined whether oxidative DNA damage was randomly distributed or whether it was localized in specific sequences in either the nuclear or mitochondrial genomes. The DNA damage-specific histone, gamma-H2AX, was detected immunohistochemically in alveolar wall cells in lung tissue from COPD patients but not control subjects. A PCR-based method was used to search for oxidized purine base products in selected 200 bp sequences in promoters and coding regions of the VEGF, TGF-β1, HO-1, Egr1, and β-actin genes while quantitative Southern blot analysis was used to detect oxidative damage to the mitochondrial genome in lung tissue from control subjects and COPD patients. Among the nuclear genes examined, oxidative damage was detected in only 1 sequence in lung tissue from COPD patients: the hypoxic response element (HRE) of the VEGF promoter. The content of VEGF mRNA also was reduced in COPD lung tissue. Mitochondrial DNA content was unaltered in COPD lung tissue, but there was a substantial increase in mitochondrial DNA strand breaks and/or abasic sites. These findings show that oxidative DNA damage in COPD lungs is prominent in the HRE of the VEGF promoter and in the mitochondrial genome and raise the intriguing possibility that genome and sequence-specific oxidative DNA damage could contribute to transcriptional dysregulation and cell fate decisions in COPD.Keywords: DNA damage, VEGF hypoxic response element, mtDNA, COPD

The 1973 Trojan survey of a region close to the preceding Lagrangian point is discussed in this paper. This region consists of four overlapping fields and one field for the photometric calibration, centered on Selected Area 68. Orbital elements of most of the 1504 asteroids found have been published in the Minor Planet Circulars. Among these are 5 Hungaria-type asteroids, 12 of the Hilda type and 55 Trojans. The accuracy of the orbits is somewhat lower than in the Palomar-Leiden survey of 1960, because of the shorter arc in the present investigation. A discussion of the degree of completeness of this material shows that the (photographic) magnitude interval 19.5–20.0 is only 50% complete; to obtain completeness down to magnitude 20.0 the number of found Trojans brighter than this limit has to be increased by 9. In combination with the surveys of 1960 and 1965 an improved distribution of the number of Trojans as a function of distance to the preceding Lagrangian point (L4) has been constructed. A preliminary discussion, including the result on L5, yields a value of 2.0 for the ratio of Trojans around L4 and L5, along the ecliptic. The L5 Trojans appear to be deficient in orbits of small inclination. Taking this into account we find this ratio again to be 2.0 for the total Trojan clouds around L4 and L5. Possibly, however, high-inclination Trojans were systematically overlooked in this survey. If this would be true, the ratio N (L4)/ N (L5) is increased to 2.3. The uncertainty of these numbers can be estimated to be ±0.5.

The synthesis rates of ribonuclease III (RNase III) and Era proteins are relatively low, and expression of the era gene is translationally coupled with expression of the rnc gene. Expression of both genes is negatively controlled by RNase III itself. We have constructed plasmids that overproduce RNase III and/or Era proteins under the control of the lambda PL promoter. A plasmid with the rnc gene under PL control expresses RNase III at levels greater than 40% of total cellular protein. Another plasmid with the era gene under PL control and a modified translation-initiation signal produces up to 80% of total cell protein as Era. Each protein has been purified using simple and rapid procedures. Purified RNase III protein specifically processes mRNA transcripts containing known RNase III sites. The purified Era protein binds GDP and GTP and has GTPase activity. Kinetic analysis shows that one molecule of GTP or GDP is bound/Era peptide with a Kd of 5.5 microM for GTP binding and 1.0 microM for GDP binding. The Km of the Era GTPase is 9.0 microM, and the maximum catalyzed rate of GTP hydrolyzed/min/mol of Era protein at 37 degrees C is 9.8 mmol.

With 60 per cent of the steam-boiler horsepower of the United States in locomotives, which operate under widely diverse conditions and with waters of many compositions, this field offers extensive opportunity for the study of embrittlement cracking. The authors present a record of the experience of The Chesapeake & Ohio Railway with intercrystalline cracking as representative of the more recent work being done by the railroads to combat this form of metal deterioration. The paper deals with the investigation of various inhibitors, covering a period from 1933 to the present time, based on extensive testing with the embrittlement detector applied to locomotives in service. It was found that sodium sulphate does not prevent or even delay embrittlement cracking; that waste sulphite liquor will prevent cracking of detector specimens on many boiler waters, failing in but few cases; and that sodium nitrate has essentially eliminated cracking of detector specimens on the entire system of The Chesapeake & Ohio Railway.

Records indicate that the first concerted study for improvement of water quality used on American railroads was conducted by the American Master Mechanics Association, which was the forerunner of the present Mechanical Division of the American Railway Association. The study was started in 1 870 and continued for three years with the resulting conclusion that the annual damage done to locomotive boilers by the average waters available was $750 per locomotive, made up of additional boiler repairs, $360, additional coal (at $2.50 per ton), $340, and additional boiler washing, $50. As an indication that these figures were not badly out of line, recent investigations by the Water Service Committee of the American Railway Engineering Association have shown that the present cost of damage caused by uncorrected poor quality waters will vary from $1,500 to $4,000 per locomotive per year which is comparable with the increase in cost of locomotives, material, and labor over the 1870 prices. The boiler damage in the period of the American Master Mechanics Association of 1870 consisted principally of scale with resultant boiler leaking and short life of flues and fire box. The proper remedy was reported and recommended, but due to the greater attention being given to railroad expansion and less consideration to features involving operating economies, action was delayed and it was not until 1891 that the Union Pacific was reported to have built the first water treating plant used on any American railroad for the purpose of delivering water to roadside tanks fit for boiler use. Additional plants were installed on the Southern Pacific and in Ohio in 1896. The results from these plants must have been satisfactory as the period beween 1900 and 1905 witnessed considerable activity in this field. The Water Service Committee report published in the Pro

1. 1. Diamond is not colored by the penetrating (beta and gamma) radiation from radium salt in glass tubes 0.5 to 1 mm. thick. 2. 2. Diamond is always colored green by direct radiation, either from high-grade radium salts or from the gas, radium emanation. No exceptions were met in more than thirty specimens. 3. 3. The green color is apparently light-permanent, but can be removed by heating to a dull red. 4. 4. Yellow and brown diamonds are also colored green, but are restored by heating to their original colors. In the case of large deep brown stones the color masking by green was not complete. 5. 5. Recutting the crystal after coloring removes the green color. This is possibly a heat effect during cutting, and does not prove that the coloring is merely a surface effect. 6. 6. The depth of penetration of the color zone could not be determined. If it is an alpha-ray effect, it would be limited by the range of alpha rays in diamond, about 0.001 inch. But reflection of green light from the highly polished surface interferes with optical observations. 7. 7. Radiation by emanation is more effective than by salts, owing to the greater absorption of alpha rays by the salt itself. 8. 8. In some cases in emanation, but never in salt, the radiation resulted in the production of “carbon spots” in the interior of the crystal. The black spots are probably a form of carbon, the nature of which was not definitely determined. They can be removed by prolonged heating in a blast flame. 9. 9. The “carbon spots” are undoubtedly beyond the range of alpha rays in the crystals, which suggests that the color may be penetrating also and that both are produced by some form of secondary radiation more penetrating than alpha rays.

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.