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In autumn, the dissolved organic matter (DOM) contribution of leaf litter leachate to streams in forested watersheds changes as trees undergo resorption, senescence, and leaf abscission. Despite its biogeochemical importance, little work has investigated how leaf litter leachate DOM changes throughout autumn and how any changes might differ inter- and intraspecifically. Since climate change is expected to cause vegetation migration, it is necessary to learn how changes in forest composition could affect DOM inputs via leaf litter leachate. We examined changes in leaf litter leachate fluorescent DOM (FDOM) from American beech (Fagus grandifolia Ehrh.) leaves in Maryland, Rhode Island, Vermont, and North Carolina and from yellow poplar (Liriodendron tulipifera L.) leaves from Maryland. FDOM in leachate samples was characterized by excitation-emission matrices (EEMs). A six-component parallel factor analysis (PARAFAC) model was created to identify components that accounted for the majority of the variation in the dataset. Self-organizing maps (SOM) compared the PARAFAC component proportions of leachate samples. Phenophase and species exerted much stronger influence on the determination of a sample's SOM placement than geographic origin. As expected, FDOM from all trees transitioned from more protein-like components to more humic-like components with senescence. Percent greenness of sampled leaves and the proportion of tyrosine-like component 1 were found to be significantly different between the two genetic beech clusters, suggesting differences in photosynthesis and resorption. Our results highlight the need to account for inter- and intraspecific variations in leaf litter leachate FDOM throughout autumn when examining the influence of allochthonous inputs to streams.

Percutaneous vertebroplasty, comprising an injection of polymethylmethacrylate (PMMA) into vertebral bodies, is a practical procedure for the stabilization of osteoporotic compression fractures as well as other weakening lesions. Cement leakage is considered to be one of the major and most severe complications during percutaneous vertebroplasty. The viscosity of the material plays a key role in this context. In order to enhance the safety for the patient, a rheometer system was developed to measure the cement viscosity intraoperatively. For this development, it is of great importance to know the proper viscosity to start the procedure determined by experienced surgeons and the relation between the time period when different injection devices are used and the cement viscosity. The purpose of the study was to investigate the viscosity ranges for different injection systems during conventional vertebroplasty. Clinically observed viscosity values and related time periods showed high scattering. In order to get a better understanding of the clinical observations, cement viscosity during hardening at different ambient temperatures and by simulation of the body temperature was investigated in vitro. It could be concluded, that the direct viscosity assessment with a rheometer during vertebroplasty can help clinicians to define a lower threshold viscosity and thereby decrease the risk of leakage and make adjustments to their injection technique in real time. Secondly, the acceleration in hardening of PMMA-based cements at body temperature can be useful in minimizing leakages by addressing them with a short injection break.

Digital ground surveys are typically conducted by total station prior to road (re)construction to obtain a three‐dimensional model of natural and man‐made features within the corridor. A study has been undertaken to ascertain whether two types of terrestrial laser scanner could meet the Main Roads Western Australia accuracy standard for digital ground survey Results indicate that the Standard can be met for surfaces and interpolated points on feature strings, such as lane markings and kerb gullies, but not for other point and linear features such as those located flush to the ground or requiring human interpretation to extract their position.

NASA’s extra-vehicular activities (EVAs) involve exposure to high energy photons while breathing 100% oxygen. Using previously verified mouse models, our laboratory is studying whether low dose irradiation under these hyperoxic conditions could lead to an increase in carcinogenic potential. To simulate the environment astronauts encounter during an EVA, enclosed chambers were constructed that allowed for mouse movement, controlled gas conditions, and uniform radiation dose delivery. Custom-built gas chambers with input/output gas valves and dividers that allowed for uniform gas flow were used to keep 6 unanesthetized mice separated while they were irradiated. The chambers were supplied with 100% oxygen or air using ball valves linked together with T-splitters. A calibrated ion chamber was used to verify the radiation dose distribution across an entire chamber. Mice were placed in the gas environments for 0.5 h, irradiated with a 10 or 18 MV photon beam from a medical linear accelerator, and left in their gas environment for 2 h post-irradiation. We irradiated 200 mice (5 different doses between 0–1000 mGy) under normoxic or 100% oxygen conditions. For the next step of this research, these mice will be euthanized 9 months post-irradiation, and lung tumors will be counted and sized to determine if hyperoxia increases the carcinogenic effect for this model.

Ventilator frequency is one of the determinants of tidal volume delivery during high-frequency ventilation. Clinicians increasingly use data on ventilator displays to inform their decisions.To measure the frequencies delivered by the Dräger Babylog 8000plus ventilator when used in high-frequency mode.Ventilator waveforms using a test lung were recorded at the full range of settings 5-20 Hz using Spectra software at 1000 Hz. The changes in frequency produced by a 1-Hz change in set frequency were calculated. Actual and displayed frequencies were compared.For settings up to 12 Hz, median (range) difference between set and delivered frequencies was 0 (-0.4 to +0.1) Hz. Above 12 Hz, delivered frequency varied by -0.3 (-1.9 to +0.3) Hz. For 1-Hz changes in frequency settings, in the range 5-12 Hz, 1-Hz changes produced a change in delivered frequency of 1.0 (0.6-1.4) Hz. Above 12 Hz, the corresponding changes were 0.7 (0-2.9) Hz. The ventilator displays the set frequency during operation rather than the delivered frequency.At 12 Hz and below, the differences between set and delivered frequencies were relatively small compared with those at 13 Hz and higher. Above 13 Hz, the difference between set and delivered frequencies was up to 2.9 Hz. Some frequency setting changes did not result in a change in delivered frequency.

The crystal and molecular structure of the title compound, 1,2,3,4-tetraphenyl-5-(4-pyridyl)bicyclo[2.2.1]hept-2-en-7-one, C36H27NO, (I), has been determined to confirm stereochemistry and investigate the potential inclusion behavior.

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top-sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift-out. The lift-out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top-cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is approximately 300 nm for top-cut sections versus approximately 100 nm for electron-transparent sections.

The acute toxicity, pharmacokinetics and hypoxic cytotoxicity of RSU-1069 were investigated using the subcutaneous (sc) rat 9L tumour model. The pharmacokinetics were studied after i.p. injection of RSU-1069 (20 mg kg-1 or 100 mg kg-1). For both doses, the elimination of RSU-1069 followed first-order kinetics in both plasma and unclamped tumours. After 100 mg kg-1, the peak plasma concentration of RSU-1069 was 40 micrograms ml-1; the elimination t1/2 was 39.3 +/- 11.1 min. After 20 mg kg-1, the peak plasma concentration was 3 micrograms ml-1; the elimination t1/2 was 47.8 +/- 6.3 min. In unclamped tumours, the peak concentration was 50 micrograms g-1 with an elimination t1/2 of 36.1 +/- 9.6 min for the 100 mg kg-1 dose, and 4 micrograms g-1 with an elimination t1/2 of 41.9 +/- 6.1 min for the 20 mg kg-1 dose. The tumour and plasma elimination half-times were not significantly different (P greater than 0.2) for the two doses. Clamping the tumour 30 min after administration of 100 mg kg-1 of RSU-1069 decreased the tumour elimination t1/2 to 10.9 +/- 1.4 min. After releasing the clamp, RSU-1069 returned rapidly to the unclamped tumour concentration. The unclamped tumour/plasma ratio reached a maximum of 4-6, then decreased to a constant value of about 2 for both doses, indicating that RSU-1069 accumulates in these 9L tumours. RSU-1069 kills hypoxic sc 9L cells more efficiently than oxic sc 9L cells; at a surviving fraction of 0.5, the SER was 4.8. For in vitro 9L cells, the SER was approximately 50 when the comparison was between those treated in 2.1% 0(2) and those treated in less than 7.5 x 10(-3)% 0(2); it was approximately 100 when the comparison was between those treated in 21% 0(2) and those treated in less than 7.5 x 10(-3)% 0(3). Tumours treated with RSU-1069 and clamped for various times exhibited biphasic cell-kill kinetics; at 50 mg kg-1, little additional cell kill was achieved after 40 min of clamping. Our data also indicate that RSU-1069 is 300-1000 fold more efficient than misonidazole or SR2508 for killing hypoxic sc 9L tumour cells in situ.

Complete genome sequences are available for understanding biotransformation, environmental resistance and pathogenesis of microbial, cellular and pathogen systems. The present technological and scientific challenges are to unravel the relationships between the organization and function of protein complexes at cell, microbial and pathogens surfaces, to understand how these complexes evolve during the bacterial, cellular and pathogen life cycles, and how they respond to environmental changes, chemical stimulants and therapeutics. In particular, elucidating the molecular structure and architecture of human pathogen surfaces is essential to understanding mechanisms of pathogenesis, immune response, physicochemical interactions, environmental resistance and development of countermeasures against bioterrorist agents. The objective of this project was to investigate the architecture, proteomic structure, and function of bacterial spores through a combination of high-resolution in vitro atomic force microscopy (AFM) and AFM-based immunolabeling with threat-specific antibodies. Particular attention in this project was focused on spore forming Bacillus species including the Sterne vaccine strain of Bacillus anthracis and the spore forming near-neighbor of Clostridium botulinum, C. novyi-NT. Bacillus species, including B. anthracis, the causative agent of inhalation anthrax are laboratory models for elucidating spore structure/function. Even though the complete genome sequence is available for B. subtilis, cereus, anthracis and other species, the determination and composition of spore structure/function is not understood. Prof. B. Vogelstein and colleagues at the John Hopkins University have recently developed a breakthrough bacteriolytic therapy for cancer treatment (1). They discovered that intravenously injected Clostridium novyi-NT spores germinate exclusively within the avascular regions of tumors in mice and destroy advanced cancerous lesions. The bacteria were also found to significantly improve the efficacy of chemotherapeutic drugs and radiotherapy (2,3). Currently, there is no understanding of the structure-function relationships of Clostridium novyi-NT spores. As well as their therapeutic interest, studies of Clostridium noyii spores could provide a model for further studies of human pathogenic spore formers including Clostridium botulinum and Clostridium perfringens. This project involved a multi-institutional collaboration of our LLNL group with the groups of Prof. T.J. Leighton (Children's Hospital Oakland Research Institute) and Prof. B. Vogelstein (The Howard Hughes Medical Institute and the Ludwig Center for Cancer Genetics and Therapeutics at The John Hopkins Sidney Kimmel Comprehensive Cancer Center).

Vacuoles of high purity were isolated from the leaves of the halophyte Suaeda maritima (L.) Dum. The relative compositions of phospholipids, phytosterols, and fatty acids in the tonoplast membrane were determined and membrane fluidity was assessed by electron spin resonance. The characteristics of the tonoplast were consistent with minimizing passive permeability to NaCI. The phospholipid: protein ratio (1.1: 1.0) was higher than that recorded in other membrane preparations, including vacuoles from beetroot storage material, commensurate with the low density (1.05 g cm−3) of the S. maritima tonoplast. The tonoplast fatty acids were highly saturated and dominated by n-hexadecanoic acid and n-octadecanoic acid. Phytosterols identified by gas chromatography were cholesterol, campesterol, stigmasterol, and β-sitosterol. Cholesterol was a trace percentage in protoplasts, but comprised 30% of the tonoplast sterols. Semi-quantitative analysis by chromatography on silica gel revealed an enrichment in the tonoplast of glycolipid which was not accounted for as chloroplast contamination. The fluidity of the tonoplast, determined by electron spin resonance, was lower than the protoplasts, consistent with the high degree of saturation of the fatty acid chains. The relevance of the lipid composition of the tonoplast to its central role in ion compartmentation within the halophyte cell is discussed.

The crystal and molecular structure of the title compound, 1,2,3,4-tetraphenyl-5-(4-pyridyl)bicyclo[2.2.1]hept-2-en-7-one, C36H27NO, (I), has been determined to confirm stereochemistry and investigate the potential inclusion behavior.

Sicel Technologies has developed an in vivo detector for /sup 14/C labeled compounds. Initial studies in rats have been completed in which labeled 2-deoxyglucose and 5-fluoruracil have been monitored dynamically in both tumors and normal tissue. These results are discussed in the context of monitoring tumor metabolism and chemotherapeutic drug uptake.

The role of bone marrow transplantation (BMT) in first remission of children with high-risk acute lymphoblastic leukemia (ALL) remains unclear. There were 3676 patients (aged 1 to 15 years) entered into the United Kingdom (UK) Medical Research Council (MRC) trials UKALL X and XI from 1985 to 1997. Of these patients, 473 patients (13%) were classified as very high (VH) risk and were eligible for a transplantation from a matched histocompatible sibling donor (MSD). We tissue-typed 286 patients; 99 patients had a matched related donor, and 76 patients received transplantations. Additionally, 25 children received transplantations from a matched unrelated donor (MUD) despite trial guidelines for MSD transplantations only. The median time to transplantation was 5 months (range, 2 to 19 months), and the median follow-up was 8 years. The 10-year event-free survival (EFS) adjusted for the time to transplantation, diagnostic white blood cell (WBC) count, Ph chromosome status, and ploidy was 6. 0% higher (95% confidence interval (CI), -10.5% to 22.5%) for 101 patients who received a first-remission transplantation (MSD and MUD) than for the 351 patients treated with chemotherapy (transplantation, 45.3%, vs chemotherapy, 39.3%). The transplantation group had fewer relapses (31%) compared to relapses in the chemotherapy group (55%); however, the transplantation group had more remission deaths (18%) compared to remission deaths in the chemotherapy group (3%). In contrast the adjusted 10-year EFS was 10. 7% higher (95% CI, -2.6% to 24.0%) for patients without a human leukocyte antigen (HLA)-matched donor than for those patients with a donor (no donor, 50.4%, vs donor, 39.7%). In conclusion, for the majority of children with VH-risk ALL, the first-remission transplantation has not improved EFS.

For several years, we have concentrated our efforts on validating the use of radiation-induced DNA strand breaks and DNA-protein crosslinks to assess the oxygenation status of tumors and normal tissues. We have demonstrated that (1) the oxygen dependence of strand break formation is identical to that of radiation-induced cell killing; (2) the oxygen dependence of DNA-protein crosslink formation is the mirror image of that of radiation-induced cell killing; and (3) the formation of these radiation-induced DNA lesions is predominantly dependent on the oxygen concentration near the DNA and is independent of the cell type, metabolic status, proliferative status, pH of the surrounding environment, and composition or properties of the proteins tightly associated with the DNA. In the present study, the hypoxic fraction of three experimental tumors was estimated using our assay of radiation-induced DNA damage. The average hypoxic fraction of a large number of tumors estimated with this assay of radiation-induced DNA damage for (1) WiDR human colorectal carcinoma xenografts (40.8 +/- 4.2%), (2) 66 mouse mammary adenocarcinoma tumors (41.8 +/- 3.1%), and (3) subcutaneous tumors grown from 9L rat brain tumor cells (95% CI =-8.2-4.2%) was not statistically different from that of a large number of tumors measured for each of these tumor models by the paired survival curve method (38.3 +/- 6. 3%, 28.9 +/- 5.5%, 95% CI = 2.2-4.4%, respectively). When the hypoxic fraction measured by the alkaline elution method on one half of an individual tumor was compared to that measured by the paired survival curve method on the other half of the same tumor, no statistical correlation was found for either 66 or WiDR tumors. Although this assay of radiation-induced DNA damage can be used effectively in the laboratory to answer a number of important questions about the oxygenation status of animal tumors and normal tissues, failure to reliably estimate the hypoxic fraction of individual tumors and technical considerations make it unlikely that the assay can be used in the clinic to estimate the hypoxic fraction of human tumors.

This article provides practitioners with meal plan samples at four calorie levels as a guide for planning athletes' food choices. Many good and practical publications are available for trainers and dietitians who assist athletes in their nutritional plans. The two primary needs of people with increased physical activity are increased energy and fluid intakes. As illustrated in this article, these energy needs can be met fairly easily at various calorie levels with a meal plan. For individual requirements, athletes should consult with health care professionals for calorie and fluid requirements.

It has been demonstrated that the yield of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) appears to depend predominantly on the oxygen concentration in the microenvironment around the DNA of mammalian cells (Radiat. Res. 142, 163-168, 1995). Consequently, these DNA lesions have the potential to be used to detect hypoxic cells or estimate the hypoxic fraction of solid tumors. Although it has been demonstrated that physiological factors (e.g. pH, temperature, nutrient depletion, etc.) have no influence on the induction and repair of both DNA strand breaks and DPCs in irradiated oxygenated cells (Radiat. Res. 140, 321-326, 1994), there are no data to suggest how these physiological factors influence the induction and repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic cells. In the present study, the influence of pH (6.6-7.3) and nutrient depletion on the formation and/or repair of radiation-induced DNA strand breaks and DPCs in partially hypoxic (O2 concentration in the gas phase of 2.0-8.0%) or fully hypoxic (O2 concentration in the gas phase of 0.3-0.4%) populations of exponential (day 3) and plateau-phase (day 5) 9L rat brain tumor cells in culture was determined. Each population of cells was irradiated at 37 degrees C with 15 Gy and trypsinized at 4 degrees C after one half-time of strand break repair at 37 degrees C, and the DNA damage was measured using our alkaline elution technique, with or without proteinase K (PK) in the lysis solution. An analysis of the DNA-protein crosslink factor as a function of the oxygen concentration and a statistical comparison of the single-strand scission factor measured at eluted fraction 7 (SSSF7), with or without PK in the lysis solution, indicate that pH and nutrient depletion do not significantly influence the formation and/or repair of DNA strand breaks and DPCs in irradiated partially hypoxic or fully hypoxic 9L cells. The data also demonstrate that the DNA lesions produced in irradiated partially hypoxic cells will cause the hypoxic fraction or fractional hypoxic volume to be overestimated by this DNA damage assay, not underestimated. However, this potential overestimation of the hypoxic fraction or fractional hypoxic volume should not limit the usefulness of this DNA damage assay in most laboratory studies, because it will take the contribution of a large cohort of partially hypoxic cells to overcome the dominance of the signal from a relatively small cohort of fully hypoxic cells.

We wished to test the hypothesis that the non proline cis to trans isomerization of the peptide bond at position 167 in the S. aureus beta-lactamase PC1 exerts a significant controlling effect on the folding pathway of this enzyme. The previous data presented in support of this hypothesis could not rule out the effect of factors unrelated to non-proline cis/trans isomerization. We have used the plasmid pET9d to direct soluble overproduction of the S. aureus beta-lactamase PC1 and a site-directed mutant (Ile 167 to Pro) in Escherichia coli. Following purification the proteins were subjected to a comparative analysis of the kinetics of unfolding and refolding using the techniques of near- and far-UV circular dichroism spectroscopy and fluorescence spectroscopy in conjunction with "double-jump" experiments. Results show that the fully-unfolded I167P mutant enzyme retains 20% of molecules in a fast-refolding form and that slower-refolding molecules fold faster than the recombinant wild-type enzyme. The final stage of folding involves folding of the omega-loop into a conformation essential for enzymatic activity. In support of the original hypothesis, the folding of this omega-loop is rate limited by the isomerization of the Glu 166-Ile 167 peptide bond.

C25H13N7O2, monoclinic, C12/c1 (no. 15), a = 24.404(3) Å, b = 6.487(1)Å, c = 13.487(2)Å, β = 108.29(1)°, V = 2027.4Å3, Z = 4, Rgt(F) = 0.038, wRref(F2) = 0.099, T = 173 K.

Recently, we demonstrated that the oxygen dependence of the formation of DNA-protein crosslinks (DPCs) in irradiated mammalian cells measured by the alkaline elution technique is the mirror image of the oxygen dependence of radiation-induced cell killing. Consequently, these radiation-induced DPCs could be used to detect hypoxic cells or estimate the hypoxic fraction of cells in solid tumors. Although several techniques, including alkaline elution, gas chromatography/mass spectrometry (GC/MS) and nitrocellulose filter binding, have been used to measure radiation-induced DPCs, the published reports suggest that the characteristics of these DPCs may depend on both the type of sample irradiated (cellular compared to model systems, oxygenated compared to hypoxic, etc.) and the technique used to measure these radiation-induced DPCs. In the present study, the radiation-induced DPCs measured by our alkaline elution technique with and without proteinase K in the lysis solution were characterized by studying the dependence of their formation on temperature in hypoxic rat 9L brain tumor cells. Exponentially growing 9L rat brain tumor cells were rendered hypoxic at 4 degrees C or at 37 degrees C and then irradiated with either 7.5 Gy or 15 Gy. The cells were trypsinized at 4 degrees C, either immediately after the irradiation or after one half-time of strand break repair at 37 degrees C. The results demonstrated that the radiation-induced DPCs produced in 9L cells under hypoxic conditions, measured by our alkaline elution technique after low to moderate radiation doses, required metabolism for their formation, unlike the radiation-induced DPCs reported by others using the GC/MS or nitrocellulose filter binding technique.

Using a variety of techniques, several laboratories have recently demonstrated the feasibility of using radiation-induced DNA strand breaks and/or DNA-protein crosslinks (DPCs) to detect and/or quantify hypoxic cells in tumors and normal tissues. However, if strand breaks and/or DPCs are to be used to estimate the hypoxic fraction or the fractional hypoxic volume of tumors and normal tissues, their formation as a function of the oxygen concentration near the DNA must be relatively independent of the biological properties of these cells. In the present study, the shape of the oxygen dependence curves and the K(m) values for radiation-induced strand breaks and DPCs were measured by alkaline elution for proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66. The sigmoidal shape of the oxygen dependence curves, the K(m) for strand breaks (approximately 0.027 mM) and the K(m) for the formation of DPCs (approximately 0.020 mM) were identical for the P and Q cells of line 66. Consequently, the proliferative status of these tumor cells had no measurable influence on the oxygen-dependent formation of radiation-induced strand breaks and DPCs. In addition, the percentage of the DNA retained on the filters after approximately 24 ml of elution without proteinase K in the lysis solution, a parameter equal to the sum of the strand breaks and DPCs that has been shown to be proportional to the percentage of hypoxic cells in the sample, was not significantly different for fully oxygenated or fully hypoxic populations from five tumor cell lines that varied in species, site of origin, proliferative status and/or properties of the proteins which are intimately associated with their DNA. These data indicate that the formation of radiation-induced strand breaks and DPCs depends predominantly on the oxygen concentration in the microenvironment around the DNA, and only minimally on the biological properties of the cells.

sigma 1 and sigma 2 receptors have been shown to exist in a number of rodent and human tumor cell lines. Although their expression is heterogeneous and their function is unknown, sigma receptors have been proposed as potential targets for diagnostic tumor-imaging agents. In this study, the density of sigma 2 receptors in proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66, was examined. Scatchard analyses of sigma 2 receptors were performed on membrane preparations of 66 P cells from 3-day cultures and 66 Q cells from 7-, 10-, and 12-day cultures. The Scatchard studies revealed that 66 P cells had approximately 10 times more sigma 2 receptors/cell than the 66 Q cells from 10-day cultures. Although97% of the cells were quiescent after 7 days in culture, the maximum differential in the sigma 2 expression between 66 P and 66 Q cells was not attained until these cells had been in culture for 10 days. These data suggest that ligands labeled with positron-emitting or single photon-emitting radionuclides, which selectively bind sigma 2 receptors, have the potential to noninvasively assess the proliferative status of human breast tumors.

The induction of base damage products in gamma-irradiated DNA, hydrated between 2.5 and 32.8 moles of water per mole of nucleotide (tau), was investigated using the gas chromatography/mass spectrometry-selected ion monitoring technique. In general, the yields of the measured base damage products were found to be dependent on the extent of the hydration when the DNA was irradiated under nitrogen. At low hydrations (tauor = 13), the highest yields of the measured products were found for 7,8-dihydro-8-oxo-guanine, 5,6-dihydrothymine and, to a lesser extent, 2,6-diamino-4-oxo-5-formamidopyrimidine, products which are consistent with the base radicals found in low-temperature ESR studies. At higher hydrations (tauor = 13), changes in DNA conformation and an increase in the attack of bulk water radicals on DNA play a significant role in the formation of radiation-induced DNA base damage products. Additional findings in our study include: (1) the sum of the yields of the products formed from electron-loss centers is greater than the sum of the yields of the products formed from electron-gain centers, indicating that there might be other electron-gain products which have not been identified; (2) the combined yield for the base damage products and the release of unaltered bases at tauor = 13 is constant, implying that radiation damage in the tightly bound water molecules of the primary hydration layer causes DNA damage (quasi-direct effect) that is similar to the damage caused by direct ionization of the DNA (direct effect); and (3) the yields of the individual base damage products that were formed from electron-loss centers can be modeled on the basis of both the known reactions that lead to the formation of the initial charged base radicals in irradiated DNA, and the known reactions that involve the conversion of these initial DNA radicals into their respective nonradical end products.

A new class of video rate imagers based on back-illuminated and thinned CCDs is available that shows promise to replace conventional image intensifiers for most military, industrial, and scientific applications. Thinned, back-illuminated CCDs (BCCDs) and electron-bombardment CCDs (EBCCDs) offer low light level performance superior to conventional image intensifier coupled CCD (ICCD) approaches. These new, high performance devices promise to expand the fields of science, provide high contrast, high resolution, low light level surveillance imaging, and make nighttime pilotage safer for military aviators. This paper presents experimental data which illustrates how responsivity, gain, and modulation transfer function (MTF) determine the low light imaging capability, the 'target of interest' signal to noise ratio (SNR) of each of these types of sensors. High SNR and MTF make BCCDs the imager of choice under moderately low light levels and EBCCDs the imager of choice under extremely low light level conditions.

Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicted on both published and unpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-induced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37 degrees C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the Km value for radiation-induced strand breaks and DPCs were similar when 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4 degrees C and nonideal gas-liquid equilibrium conditions at 37 degrees C. At 4 degrees C under ideal gas-liquid equilibrium conditions, the Km for the formation of strand breaks was approximately 0.0045 mM, and the Km for radiation sensitivity was approximately 0.005 mM. A similar comparison for the formation of DPCs at 4 degrees C could not be made, because the efficiency of the formation of DPCs was much lower at 4 degrees C than at 37 degrees C. At 37 degrees C under nonideal gas-liquid equilibrium conditions, the apparent Km for the formation of strand breaks and radiation sensitivity was approximately 0.032 mM, and the Km for the formation of DPCs was approximately 0.02 mM. The data for strand breaks are in agreement with the published data of Chapman et al. (Int. J. Radiat. Biol. 26, 383-389, 1974), and the data for DPCs are in agreement with the unpublished data of Meyn (personal communication). These results support the suggestion that measurements of radiation-induced strand breaks and/or DPCs may be used to detect hypoxic cells and estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues.

Detection and quantification of hypoxic cells in solid tumors is important for many experimental and clinical situations. Several laboratories, including ours, have suggested that assays which measure radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elution assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must not be formed to any great extent in irradiated oxygenated cells, and the formation and repair of strand breaks and DPCs in oxygenated cells must not be modified appreciably by physiological factors (e.g. temperature, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In one set of experiments, oxygenated 9L cells in tissue culture, subcutaneous 9L tumors and rat cerebella were irradiated with doses of 15 or 50 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated exponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose of 15 Gy at 37 or 20 degrees C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of strand breaks or the formation of DPCs in oxygenated 9L cells. These results support the use of this DNA damage assay for the detection and quantification of hypoxic cells in solid tumors.

years. Early single anode and multianode MCPIPIVfls were described by Boutot & Pietri' andCatchpole & Johnson2, respectively. Some recent advances that are being made are describedin this paper. Compact and rugged photomultiplier tubes (PMTs) are becoming increasinglyimportant as electro-optical sensors for land and airborne tactical military systems. They havethe ability to detect individual photons over a broad spectral range, from the uv to the near-frregions. A multianode MCP/PMT has been developed with a lOxlO array of parallel signal

Photomultiplier tubes (PMTs) making use of microchannel plate (MCP) electron multiplier assemblies are being developed. Bothsingle anode and multianode designs are available having active diameters of 18mm, 25mm and 40mm. A variety of spectralsensitivities is available in all sizes, and Blue GaAs, GaAs or InGaAs photocathodes can be provided in the 18mm active diameter size. Pulse height distributions having full-width half-maximum values of 59 % and peak-to-valley ratios of 10 have been achieved. 1. INTRODUCTION Photomultiplier tubes are being developed that make use of MCP electron multiplier assemblies. These MCP/PMTs are muchsmaller than PMTs having discrete dynode electron multipliers. A wider range of photocathode sensitivities are available; theyoffer a higher speed of response, and they have excellent pulse-height distribution for photon-counting. Also, two-dimensionalanode patterns can be used for readout of up to 140 or more separate anodes in a 40mm active diameter. Electron gain values

It is well known that the type and quantity of DNA damage produced by ionizing radiation depend on the oxygen concentration around the DNA. For example, in irradiated mammalian cells, both a decrease in the DNA strand break efficiency and the induction of DNA-protein crosslinks (DPCs) occur as the extracellular oxygen concentration is decreased below 1%. In the study reported here, the feasibility of estimating the hypoxic fraction of irradiated tumors and normal tissues was investigated by measuring the single-strand scission factor, the DNA-protein crosslink factor, and the amount of DNA remaining on polycarbonate filters after elution with approximately 24 ml of tetrapropylammonium hydroxide at pH 12.3 without proteinase K (PK) in the lysis solution. In anesthetized air-breathing Fisher 344 rats, no radiation-induced DPCs were detected in either cerebellar neurons or cells of subcutaneous (sc) 9L tumors when the DNA was assayed at approximately one half-time of repair after dosesor = 15 Gy. Within 10 min after anesthetized rats were killed, the maximum decrease in the radiation-induced strand break efficiency and the maximum formation of radiation-induced DPCs occurred in both cerebellar neurons and sc 9L tumors. When irradiated cerebellar neurons or sc 9L tumor cells from air-breathing and dead rats were mixed to simulate hypoxic fractions of 0, 10, 25, 50, 75, and 100%, only the percentage of the DNA retained on the filter after approximately 24 ml of elution without PK in the lysis solution was a linear function of the simulated hypoxic fraction after doses of both 15 and 2 Gy. At 15 Gy, the linear function was identical for 9L cells in tissue culture, sc 9L tumor cells, and cerebellar neurons. In addition, the slope, but not the intercept, of the linear function appeared to be independent of dose from 2 to 15 Gy. Consequently, if the dose and the amount of strand break repair are kept relatively constant, the linear function appears to depend primarily on radiation chemistry events, rather than the biological properties of the irradiated cells. Moreover, the data suggest that this assay can measure a hypoxic fraction ofor = 10% after a conventional radiotherapy dose of 2 Gy, provided sufficient material is available for analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Induced expression of the HSP70 gene in 3T3 and SV-3T3 cells was monitored by measurements of the synthesis of HSP70 and of the cellular contents of both HSP70 and its mRNA. The presence of betaine (N-trimethylglycine) at concentrations of 2.5-25 mM decreased the induction of HSP70 gene expression caused by incubation of 3T3 and SV-3T3 cells in hypertonic (0.5 osM) medium. This effect was accompanied by an enhancement of SV-3T3 cell adaptation, assayed by colony formation, to the hyperosmotic conditions. In contrast, the presence of betaine did not affect HSP70 gene expression induced in these cells by heat shock. After 6 h incubation with 25 mM betaine under hypertonic (0.5 osM) conditions the intracellular concentration of betaine in SV-3T3 cells was about 195 mM, compared with about 70 mM under isotonic (0.3 osM) conditions. Hence, with this concentration of extracellular betaine, the marked increase in the accumulation of betaine within the cells presumably counteracts the imposed osmotic pressure and eliminates the signal that otherwise initiates increased expression of the HSP70 gene.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.

To determine whether the half-times (T1/2) of the DNA repair processes measured by alkaline elution increased in a dose-dependent manner, exponentially growing 9L/Ro rat brain tumor cells were irradiated with doses of 15-50 Gy, and their DNA repair kinetics was measured by alkaline elution. At 15 Gy, the DNA repair kinetics was biphasic with the fast phase having a T1/2 approximately 6 min and the slow phase having a T1/2 approximately 42 min. As the dose was increased to 50 Gy, the fast-phase T1/2 remained at approximately 6 min, but the slow-phase T1/2 increased to approximately 87 min. Although a dose-dependent increase in the T1/2 of the slow phase of DNA repair (saturation) was measured by alkaline elution, both the absolute value of the slow-phase T1/2 and the dependency of the slow-phase T1/2 on dose were less than those measured by alkaline sucrose gradient sedimentation in zonal rotors with slow reorienting gradient capability. Thus these two techniques appear either to depend on different hydrodynamic properties of the DNA or to have different coefficients of dependency for the same hydrodynamic properties of the DNA. The lower sensitivity for detection of the dose dependency of DNA repair makes it unlikely that the alkaline elution technique will be useful for quantitatively relating the shape of mammalian cell survival curves to the doses at which saturation of a DNA repair process occurs.

When poly(dA), poly(dA-dT), and salmon testis DNA were gamma-irradiated under nitrogen, the major deoxyadenosine damage product (excluding liberated adenine) was identified as the alpha-anomer of deoxyadenosine. The yields of alpha-deoxyadenosine from poly(dA), poly(dA-dT), and salmon testis DNA irradiated with a dose of 500 Gy under anoxic conditions were 1.5, 1.3, and 1.3%, respectively. No alpha-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the alpha-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.

Using a variety of techniques, several laboratories have recently demonstrated the feasibility of using radiation-induced DNA strand breaks and/or DNA-protein crosslinks (DPCs) to detect and/or quantify hypoxic cells in tumors and normal tissues. However, if strand breaks and/or DPCs are to be used to estimate the hypoxic fraction or the fractional hypoxic volume of tumors and normal tissues, their formation as a function of the oxygen concentration near the DNA must be relatively independent of the biological properties of these cells. In the present study, the shape of the oxygen dependence curves and the K(m) values for radiation-induced strand breaks and DPCs were measured by alkaline elution for proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66. The sigmoidal shape of the oxygen dependence curves, the K(m) for strand breaks (approximately 0.027 mM) and the K(m) for the formation of DPCs (approximately 0.020 mM) were identical for the P and Q cells of line 66. Consequently, the proliferative status of these tumor cells had no measurable influence on the oxygen-dependent formation of radiation-induced strand breaks and DPCs. In addition, the percentage of the DNA retained on the filters after approximately 24 ml of elution without proteinase K in the lysis solution, a parameter equal to the sum of the strand breaks and DPCs that has been shown to be proportional to the percentage of hypoxic cells in the sample, was not significantly different for fully oxygenated or fully hypoxic populations from five tumor cell lines that varied in species, site of origin, proliferative status and/or properties of the proteins which are intimately associated with their DNA. These data indicate that the formation of radiation-induced strand breaks and DPCs depends predominantly on the oxygen concentration in the microenvironment around the DNA, and only minimally on the biological properties of the cells.

Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicated on both published and nonpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-produced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37{degrees}C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the K{sub m} value for radiation-induced strand breaks and DPCs were similar whenmore » 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4{degrees}C and nonideal gas-liquid equilibrium conditions at 37{degrees}C. At 4{degrees}C under ideal gas-liquid equilibrium conditions, the K{sub m} for the formation of strand breaks was approximately 0.0045 mM, and Km for radiation sensitivity was approximately 0.005mM. A similar comparison for the formation of DPCs at 4{degrees}C could not be made, because the efficiency of the formation of DPC was much lower at 4{degrees}C than at 37{degrees}C. 30 refs., 3 figs.« less

Detection and quantification of hypoxic cells in solid tumors is important for many experimental and clinical situations. Several laboratories, including ours, have suggested that assays which measure radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elution assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must not be formed to any great extent in irradiated oxygenated cells, and the formation and repair of strand breaks and DPCs in oxygenated cells must not be modified appreciably by physiological factors (e.g., temperature, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In one set of experiments, oxygenated 9L cells in tissue culture, subcutaneous 9L tumors and rat cerebella were irradiated with doses of 15 or 50 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated exponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose ofmore » 15 Gy at 37 or 20{degrees}C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of strand breaks or the formation of DPCs in oxygenated 9L cells. These results support the use of this DNA damage assay for the detection and quantification of hypoxic cells in solid tumors. 27 refs., 5 tabs.« less

It is well known that the type and quantity of DNA damage produced by ionizing radiation depend on the oxygen concentration around the DNA. For example, in irradiated mammalian cells, both a decrease in the DNA strand break efficiency and the induction of DNA-protein crosslinks (DPCs) occur as the extracellular oxygen concentration is decreased below 1%. In the study reported here, the feasibility of estimating the hypoxic fraction of irradiated tumors and normal tissues was investigated by measuring the single-strand scission factor, the DNA-protein crosslink factor, and the amount of DNA remaining on polycarbonate filters after elution with approximately 24 ml of tetrapropylammonium hydroxide at pH 12.3 without proteinase K (PK) in the lysis solution. In anesthetized air-breathing Fisher 344 rats, no radiation-induced DPCs were detected in either cerebellar neurons or cells of subcutaneous (sc) 9L tumors when the DNA was assayed at approximately one half-time of repair after doses < or = 15 Gy. Within 10 min after anesthetized rats were killed, the maximum decrease in the radiation-induced strand break efficiency and the maximum formation of radiation-induced DPCs occurred in both cerebellar neurons and sc 9L tumors. When irradiated cerebellar neurons or sc 9L tumor cells from air-breathing and dead rats were mixed to simulate hypoxic fractions of 0, 10, 25, 50, 75, and 100%, only the percentage of the DNA retained on the filter after approximately 24 ml of elution without PK in the lysis solution was a linear function of the simulated hypoxic fraction after doses of both 15 and 2 Gy. At 15 Gy, the linear function was identical for 9L cells in tissue culture, sc 9L tumor cells, and cerebellar neurons. In addition, the slope, but not the intercept, of the linear function appeared to be independent of dose from 2 to 15 Gy. Consequently, if the dose and the amount of strand break repair are kept relatively constant, the linear function appears to depend primarily on radiation chemistry events, rather than the biological properties of the irradiated cells. Moreover, the data suggest that this assay can measure a hypoxic fraction of < or = 10% after a conventional radiotherapy dose of 2 Gy, provided sufficient material is available for analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were gamma-irradiated with 1500 rad and then immediately treated for 20 min with a 37 degree C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. Hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation isotonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damage per se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions.