Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

A silicon-based, sequential coat-and-etch process to fabricate nearly perfect substrate surfaces.

For many thin-film applications substrate imperfections such as particles, pits, scratches, and general roughness, can nucleate film defects which can severely detract from the coating's performance. Previously we developed a coat-and-etch process, termed the ion beam thin film planarization process, to planarize substrate particles up to approximately 70 nm in diameter. The process relied on normal incidence etching; however, such a process induces defects nucleated by substrate pits to grow much larger. We have since developed a coat-and-etch process to planarize approximately 70 nm deep by 70 nm wide substrate pits; it relies on etching at an off-normal incidence angle, i.e., an angle of approximately 470 degrees from the substrate normal. However, a disadvantage of this pit smoothing process is that it induces defects nucleated by substrate particles to grow larger. Combining elements from both processes we have been able to develop a silicon-based, coat-and-etch process to successfully planarize approximately 70 nm substrate particles and pits simultaneously to at or below 1 nm in height; this value is important for applications such as extreme ultraviolet lithography (EUVL) masks. The coat-and-etch process has an added ability to significantly reduce high-spatial frequency roughness, rendering a nearly perfect substrate surface.

Synthesis of peptide-oligonucleotide conjugates for chromium coordination.

The synthesis of the first peptide-oligonucleotide conjugate designed to coordinate chromium(III) is reported. The overall goal of this work is to synthesize di-deoxynucleotides tethered with chromium(III)-coordinating appendages to model chromium-DNA-protein cross-links, which are a type of DNA lesion that may be involved in chromium-induced cancers. The conjugate dGp(NHCH(2)CH(2)S-Ac-Gly-Ser-Gly-OH)G was made by coupling the peptide, ClAc-Gly-Ser-Gly-OH, and dinucleotide, dGp(NHCH(2)CH(2)SH)G, through a thioether moiety. The conjugate was characterized by HPLC and mass spectrometry. Previously reported methods for small-scale solid-phase synthesis of peptides and dinucleotide were unsuitable; therefore, gram-scale solution-phase methods were developed. We also report the gram-scale syntheses of two other serine-containing peptides, ClAc-betaAla-Ser-Gly-OH and ClAc-Ser-Gly-OH, and three histidine-containing peptides, ClAc-Gly-His-Gly-OH, ClAc-betaAla-His-Gly-OH, and ClAc-His-Gly-OH. The synthesis and characterization of chromium-containing peptide-oligonucleotide conjugates will ultimately help us to understand chromium-DNA interactions at a molecular level, which is necessary before we can determine how chromium causes cancer.

The role of chromium(V) in the mechanism of chromate-induced oxidative DNA damage and cancer.

The role that high valent chromium intermediates play in the oxidative DNA damage produced by the human carcinogen chromate Cr(VI) is of increasing interest for establishing a mechanism of genotoxicity and mutagenicity for this metal. In this review, the authors summarize experimental evidence for the formation of high valent chromium complexes (primarily the +5 oxidation state) and radical species from the reductive metabolism of Cr(VI). A case is made for a direct- or metal-mediated pathway by high valent chromium to initiate oxidative DNA damage, although the role of radical species in this oxidative process cannot be ruled out.

Is chromium a trace essential metal?

If chromium is an essential metal it must have a specific role in an enzyme or cofactor, and a deficiency should produce a disease or impairment of function. To date, no chromium-containing glucose tolerance factor has been characterized, the purpose of the low-molecular-weight chromium-binding protein is questionable, and no direct interaction between chromium and insulin has been found. Furthermore, chromium3+ is treated like the toxic metals arsenic, cadmium, lead and mercury in animals. Chromium3+ may be involved in chromium6+-induced cancers because chromium6+ is converted to chromium3+ in vivo, and chromium3+ is genotoxic and mutagenic. Although there is no direct evidence of chromium deficiencies in humans, dietary supplements exist to provide supraphysiological doses of absorbable chromium3+. Chromium3+ may act clinically by interfering with iron absorption, decreasing the high iron stores that are linked to diabetes and heart disease. If so, this would make chromium3+ a pharmacological agent, not an essential metal.

Intermediates produced in the reaction of chromium(VI) with dehydroascorbate cause single-strand breaks in plasmid DNA.

Ascorbate (vitamin C) is a biological reductant of the human carcinogen chromium(VI). The product of this reaction is presumed to be dehydroascorbate. However, we have found that chromium(VI) can also react with dehydroascorbate. This reaction was monitored by UV/ visible and electron paramagnetic resonance (EPR) spectroscopies. In sodium acetate buffer at pH 3.8, the reaction of chromium(VI) and excess dehydroascorbate produced chromium(V) and chromium(IV) intermediates. At high reaction concentration, the chromium(V) intermediate formed an EPR silent dimer, which dissociated upon dilution to lower concentration. UV/ visible experiments at pH 3.8 demonstrated that manganese(II) catalyzed the disproportionation of chromium(IV) to chromium(V) and chromium(III). The ability of the reaction intermediates to induce strand breaks in pBR322 DNA was determined at pH 3.8 and pH 5.8. At pH 3.8, chromium(IV) appeared to be the major species responsible for induction of strand breaks because the time course for formation of strand breaks did not parallel that of chromium(V), and strand breaks were decreased in the presence of the chromium(IV) scavenger manganese(II). At pH 5.8, fewer strand breaks were observed; however, the time course for their formation followed that of chromium(V). There has been much effort devoted to identification of the intermediate responsible for the induction of strand breaks during reactions of chromium(VI) with biological reductants. The current results suggest that it is not a single type of species that universally produces the DNA strand breaks observed in different chromium(VI) systems and that the reactivity of intermediates will depend on the chosen experimental conditions. Understanding this variability in chromium(VI) reactions may help to resolve the conflicting results from in vitro studies that are aimed at deciphering mechanisms of chromium(VI)-induced cancers.

Chromium(III) picolinate produces chromosome damage in Chinese hamster ovary cells.

Chromium(III) complexes currently being sold as dietary supplements were tested for their ability to cause chromosomal aberrations in Chinese hamster ovary cells. Complexes were tested in soluble and particulate forms. Chromium picolinate was found to produce chromosome damage 3-fold to 18-fold above control levels for soluble doses of 0.050, 0.10, 0.50, and 1.0 mM after 24 h treatment. Particulate chromium picolinate doses of 8.0 micrograms/cm2 (corresponding to a 0.10 mM solublized dose) and 40 micrograms/cm2 (0.50 mM) produced aberrations 4-fold and 16-fold above control levels, respectively. Toxicity was measured as a decrease in plating efficiency relative to controls. The above treatments produced > or = 86% survival for all doses except 1.0 mM chromium picolinate, which produced 69 +/- 10% survival. Chromium nicotinate, nicotinic acid, and chromium(III) chloride hexahydrate did not produce chromosome damage at equivalent nontoxic doses. Damage was inferred to be caused by the picolinate ligand because picolinic acid in the absence of chromium was clastogenic. Data are evaluated in terms of their relevance to human exposure based on pharmacokinetic modeling of tissue accumulation and are discussed in terms of literature reporting toxic effects of picolinic acid.

A prediction of chromium(III) accumulation in humans from chromium dietary supplements.

It has been proposed that 90% of American's diets are deficient in the trace essential mineral chromium. Several chromium(III) dietary supplements are currently available to alleviate this deficiency. We show here that the same pharmacokinetic models that have been used to quantitate absorption of chromium(III) in humans predict that ingested chromium(III) will accumulate and be retained in human tissues for extended periods. Calculations were carried out with the popular supplement chromium picolinate as an example, but may be applied to any chromium(III) complex. Results from these calculations were compared to clinical data obtained from chromium(III) absorption/retention studies in humans. The models predict that chromium(III) can accumulate in human tissues to reach the levels at which DNA damage has been observed in animals and in vitro. The use of chromium supplements for extended periods or in excess dosages should be reevaluated in terms of these established models because the possible long-term biological effects of chromium accumulation in humans are poorly understood.

Quality of life of long-term adult survivors of autologous bone marrow transplantation.

PURPOSE/OBJECTIVES: To extend the knowledge of quality of life (QOL) of survivors of allogeneic bone marrow transplantation (BMT) to include survivors of autologous BMT. To determine the appropriateness of using a reliable, valid, allogeneic BMT QOL instrument for survivors of autologous BMT. DESIGN: Cross-sectional, descriptive, replication survey. SETTING: An autologous BMT program in a National Cancer Institute-designated comprehensive cancer center. SAMPLE: All survivors of autologous BMT (N = 37) at the center were recruited; 29 survivors participated (85% response rate). METHODS: Three mailed questionnaires: the City of Hope quantitative QOL-BMT instrument and qualitative questionnaire and an investigator-developed demographic questionnaire. MAIN RESEARCH VARIABLES: QOL, autologous BMT treatment, disease type, age, gender, employment, and date of transplant. FINDINGS: Global QOL was high (mean = 8.17 on a 1-10 scale). Most respondents experienced few long-term physical disruptions and had only mild psychological distress. Fatigue, sexuality concerns, and family distress created by the illness were the most negatively rated items. Content, face, and construct validity of the QOL-BMT instrument in the autologous BMT population were acceptable. Overall internal consistency reliability of the tool, as measured by Cronbach's alpha, was 0.82. Themes of uncertainty, concern about relapse, and pain were reported in the qualitative data but not revealed by responses to the QOL-BMT instrument. CONCLUSIONS: The majority of survivors of autologous BMT reported few physiologic disruptions and above-average QOL. The City of Hope QOL-BMT instrument had acceptable reliability and validity when adapted for survivors of autologous BMT. Addition of items related to uncertainty, pain, and concern about relapse could further strengthen its validity. IMPLICATIONS FOR NURSING PRACTICE: Most survivors of autologous BMT can expect above-average QOL. Incorporating results of QOL evaluation in the informed consent process may help BMT candidates in making the decision to undergo the procedure by providing a more complete picture of life after BMT. However, since a minority of patients will experience continued disruptions in any one of the QOL domains, healthcare providers need to conduct comprehensive follow-up evaluations to determine which patients may need referral to the specialized services of a survivor clinic.

Fever in geriatric emergency patients: clinical features associated with serious illness.

STUDY OBJECTIVE: To determine the clinical significance of fever in geriatric emergency department patients. DESIGN: Case series with follow-up. SETTING: Urban, university-affiliated community hospital. PARTICIPANTS: Consecutive patients over the age of 65 years who presented to the ED during a 12-month period with an oral temperature of 100.0 degrees F (37.8 degrees C) or higher. RESULTS: We considered the following features indicators of serious illness: positive blood culture(s), related death within 1 month of ED visit, need for surgery or other invasive procedure, hospitalization for 4 or more days, IV antibiotics for 3 or more days, and repeat ED visit within 72 hours for related condition. Four hundred eighty-nine patients were eligible for study. Of the 470 patients with complete follow-up data, 357 (76.0%) had indicators of serious illness. Clinical features found to be independently associated with serious illness included oral temperature of 103 degrees F (39.4 degrees C) or more, respiration rate of 30 or more, leukocytosis of 11.0 x 10(9)/L or more, presence of an infiltrate, and pulse of 120 or more. At least one indicator of serious illness was present in 63 of 128 patients (49.6%) with none of these independently predictive clinical features. The most common final diagnoses were pneumonia (24.0%), urinary-tract infection (21.7%), and sepsis (12.8%). CONCLUSION: Fever among geriatric ED patients frequently marks the presence of serious illness. All such patients should be strongly considered for hospital admission, particularly when certain clinical features are present. The absence of abnormal findings does not reliably rule out the possibility of serious illness.

Reduction of chromium(VI) by ascorbate leads to chromium-DNA binding and DNA strand breaks in vitro.

Chromium(VI) is a known human carcinogen which requires intracellular reduction for activation. Ascorbate (vitamin C) has been reported to function as a major reductant of Cr(VI) in animals and cell culture systems. The reaction of Cr(VI) with varying concentrations of ascorbate was studied under physiological conditions in vitro in order to determine the types of reactive intermediates produced and to evaluate the reactivity of these intermediates with DNA. Reactions of 1.8 mM Cr(VI) with 0-18 mM ascorbate at pH 7.0 in N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES; 0.10 M) and tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl; 0.050 M) buffers were studied by electron paramagnetic resonance and UV/visible spectroscopy. Cr(V) and carbon-based free radical adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were observed at 0.5 to 1 and 1 to 1 reactions of ascorbate to Cr(VI). Levels of Cr(V) were higher for reactions in HEPES buffer, and levels of carbon-based radicals were higher in Tris.HCl buffer. Levels of Cr(IV) and Cr(III) increased with increasing concentration of ascorbate in both buffers. Reaction of Cr(VI) with varying ascorbate in the presence of calf thymus DNA or pBR322 DNA resulted in Cr-DNA adducts and plasmid relaxation, respectively. Maximum binding of Cr to DNA was observed for the 1:1 reaction ratio of Cr(VI) with ascorbate in both HEPES and Tris.HCl buffers, but total Cr bound to DNA was 8-fold lower in Tris.HCl than HEPES buffer. Preincubation of Cr(VI) with ascorbate before reaction with DNA decreased Cr-DNA binding to background levels. Preincubation of Cr(III) with ascorbate resulted in only low Cr-DNA binding. Levels of Cr-DNA binding were higher with single-stranded vs double-stranded DNA. Reactions with 14C-labeled ascorbate produced no cross-linking of ascorbate to DNA. Maximum plasmid relaxation was observed for the 1:1 ascorbate to Cr(VI) ratio in both buffers; however, single-strand breaks were 2-fold higher in Tris.HCl than HEPES buffer. Reactions with plasmid in the presence of DMPO quenched formation of single-strand breaks. Interpretation of these results in light of the spectroscopic studies suggested that Cr(V) and carbon-based radicals were responsible for Cr-DNA adducts and DNA single-strand breaks, respectively.

Cell-enhanced dissolution of carcinogenic lead chromate particles: the role of individual dissolution products in clastogenesis.

Lead chromate induces chromosomal damage as a result of extracellular dissolution producing solubilized chromium and lead and we show here that the dissolution process is greatly accelerated by the presence of cells. We have sought to determine which of these ions is involved in lead chromate-induced clastogenicity. Cell-mediated extracellular dissolution of particulate lead chromate resulted in the accumulation of both solubilized chromium and solubilized lead, reaching concentrations in the extracellular medium of 15 and 1.9 microM respectively and reaching concentrations inside the cell of 2700 and 97 microM respectively. Both the extracellular and intracellular accumulation of chromium was time dependent and both the solubilized lead and chromium increased proportionately from a lower dose to a higher dose. Exposing cells to water soluble sodium chromate under conditions which produced similar time-dependent intracellular concentrations of chromium also produced a similar amount and spectrum of chromosome damage as lead chromate. In contrast, exposure to lead glutamate resulted in intracellular lead levels 438-times higher than those produced by lead chromate, but produced no chromosome damage. A higher dose of lead glutamate was weakly clastogenic, but it induced a different spectrum of chromosomal aberrations than lead chromate. Pretreatment of cells with vitamin E had no effect on the uptake of chromium, but reduced both sodium chromate- and lead chromate-induced clastogenesis by 54-93%. Vitamin E pretreatment did not affect lead glutamate-induced clastogenesis. The results of this study indicate that although lead(II) is weakly clastogenic at high doses, hexavalent chromium is the proximate clastogen in lead chromate-induced clastogenesis. Additionally, this is the first report that pretreatment of cells with vitamin E can block clastogenesis induced by particulate chromates.

Chromium(VI) reduction by ascorbate: role of reactive intermediates in DNA damage in vitro.

Reaction of chromium(VI) with one equivalent of ascorbate was studied by electron paramagnetic resonance spectroscopy in the presence of 0.10 M 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) at room temperature in 0.10 M (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES) and 0.05 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffers (pH 7.0 room temperature). Chromium(V), ascorbyl radical, and carbon-based DMPO-radical adducts were observed. A higher level of Cr(V) was observed in HEPES buffer and a higher level of the DMPO-radical adducts was observed in Tris-HCl buffer. Chromium-DNA binding studies were carried out in vitro for calf thymus DNA incubated with Cr(VI) and ascorbate in both buffers at 37 degrees C. Higher Cr-DNA binding was observed in HEPES buffer. DNA strand-break studies were carried out in vitro on pBR322 DNA incubated with Cr(VI) and ascorbate in both buffers at 37 degrees C. Higher percent nicking was observed in Tris-HCl buffer. Addition of DMPO decreased nicking levels in Tris-HCl buffer. These results suggest that free radicals are more reactive than Cr(V) in producing DNA strand breaks and that Cr(V) will react with DNA to produce Cr-DNA adducts. The fact that buffer affects the nature of the reactive intermediates produced upon reduction of Cr(VI) may be related to differences in intracellular metabolism of Cr(VI) and resulting DNA damage observed in various cell culture systems and animal tissues in vivo.

Reaction of chromium(VI) with ascorbate produces chromium(V), chromium(IV), and carbon-based radicals.

Reaction of potassium dichromate with sodium ascorbate was studied by EPR spectroscopy at room temperature, in 0.10 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), phosphate, cacodylate, and tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) buffers at pH 7.0, in the presence of 0.10 M spin trap [5,5-dimethyl-1-pyrroline 1-oxide or 2-methyl-N-(4-pyridinylmethylene)-2-propanamine N,N'-dioxide]. Chromium(V), ascorbate radical, CO2-, and other carbon-based spin trap-radical adducts were observed. Chromium(V), CO2-, and the carbon-based radicals were observed at low ratios of ascorbate to chromium, and ascorbate radical was observed at high ratios of ascorbate to chromium. The presence of Cr(IV) was detected indirectly by reaction with Mn(II) and a subsequent decrease in the Mn(II) EPR signal. More Cr(IV) was found for the higher reaction ratios of ascorbate to Cr(VI). The only buffer effect observed was a relative decrease of the Cr(V) signal in Tris.HCl vs HEPES, phosphate, and cacodylate buffers, no change in the radical adducts was observed. There was no evidence for reactive oxygen species an intermediates in this reaction. Addition of the singlet oxygen trap 2,2,6,6-tetramethyl-4-piperidone hydrochloride showed no 2,2,6,6-tetramethyl-1-piperidinyloxy radical formation. The Cr(V) species did not react with dioxygen, and dioxygen did not affect the formation of carbon-based radicals. A mechanism consistent with these observations is discussed.

Chirped multilayer coatings for increased x-ray throughput.

Chirped Mo-Si multilayer coatings, where the multilayer period is systematically varied throughout the deposition process, exhibit an increased x-ray bandwidth at normal incidence with a corresponding increase in the integrated reflectance of as much as 20% at lambda ~ 13 nm. The increased bandwidth is accompanied by a slight reduction in peak reflectance. The relation between the integrated and peak reflectance is used to determine the chirp required to optimize the x-ray throughput of a multiple-element optical system.

Multilayer mirror technology for soft-x-ray projection lithography.

Recent advances in multilayer mirror technology meet many of the stringent demands of soft-x-ray projection lithography (SXPL). The maximum normal-incidence reflectivity achieved to date is 66% for Mo/Si multilayers at a soft-x-ray wavelength of 13.4 am, which is sufficient to satisfy the x-ray throughput requirements of SXPL. These high-performance coatings can be deposited on figured optics with layer thickness control of ˜ 0.5%. Uniform multilayer coatings are required for SXPL imaging optics, for which maintaining the surface figure is critical to achieving diffraction-limited performance.

In contrast the coatings on the condenser optics will be graded to accommodate a large range of angles of incidence. Graded multilayer coatings can also be used to modify the figure of optical substrates without increasing the surface roughness. This offers a potential method for precise fabrication of aspheric imaging optics.

Ion-assisted sputter deposition of molybdenum-silicon multilayers.

X-ray multilayer (ML) structures that are fabricated by the use of magnetron-sputter deposition exhibit a degradation in structural quality as the deposition pressure is increased. The observed change in morphology is attributed to a reduced mobility of surface adsorbed atoms, which inhibits the formation of smooth, continuous layers. The application of a negative substrate bias produces ion bombardment of the growing film surface by sputtering gas ions extracted from the plasma and permits direct control of the energy density supplied to the film surface during thin-film growth. The technique supplements the energy lost to thermalization in high-pressure deposition and permits the fabrication of high-quality ML structures at elevated processing pressures. A threefold improvement in the soft-x-ray normal-incidence reflectance at 130 A results for substrate bias voltages of the order of ˜ - 150 V for Mo-Si ML's deposited at 10-mTorr Ar.