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Medieval Archaeology, 10, 81-88

In conventional data acquisition, the delay time between the firing of one source and the next is such that the energy from the previous source has decayed to an acceptable level before data associated with the following source arrives. This minimum delay time imposes constraints on the data acquisition rate. For marine data, the minimum delay time also implies a minimum inline shot interval, because the vessel’s minimum speed is limited.

(1957). An Inlaid Iron Folding Stool in the British Museum. Medieval Archaeology: Vol. 1, No. 1, pp. 39-56.

The management of renal lithiasis is a life-long proposition for most patients. Stone diseases are difficult for both patients and physicians to monitor and treat appropriately. The long-term management of metabolically active patients requires close association with a physician who is prepared to monitor stone formation, appropriate risk factors, and the progress of those risk factors over an extended time. Although this is a large economic commitment, it has been calculated that failure to carry out this responsibility is an even larger economic burden for patients and society. With aggressive risk factor management and follow-up, 95% of patients can become metabolically inactive. The long-term management of these patients, still evolving, is going to continue to require some assessment of the presence or absence of stones. The analysis of risk factors and the assessment of therapeutic maneuvers should continue to improve. It is possible that some specific index of risk factors for these patients will become readily available. Unfortunately, the efficacy of the ones currently available have not been clearly established.

Nine women with laxative abuse and predominantly ammonium urate renal calculi underwent metabolic studies to identify common chemical abnormalities and determine pathophysiology. The 24-hour urine studies demonstrated marked decreases in volume (902 cm.3), sodium (28 mEq.), citrate (116 mg.) and potassium (21 mEq.). A significant elevation in ammonium urate supersaturation was found compared to control subjects when studied by the computer model EQUIL 2. Of the patients 7 had 1 or more urine specimens positive for phenolphthalein. Gastrointestinal loss of fluid and electrolytes allowed for chronic extracellular volume depletion. Intracellular acidosis was present as judged by low urinary citrate and potassium. The fact that the ion product for ammonium urate is increased significantly compared to controls reflects the stated pathophysiological changes. Laxative abuse should be suspected whenever a woman has an ammonium urate renal calculus in sterile urine.

Single-strand breaks (SSBs) are one of the most common forms of genetic damage, arising from attack of DNA by reactive oxygen species or as intended or inadvertent products of normal cellular DNA metabolic events. Recent evidence linking defects in the enzymatic processing of nonconventional DNA SSBs, i.e., lesions incompatible with polymerase or ligase reactions, with inherited neurodegenerative disorders, reveals the importance of SSB repair in disease manifestation. I review herein the major eukaryotic enzymes (with an emphasis on the human proteins) responsible for the "clean-up" of DNA breaks harboring 3'- or 5'-blocking termini, and the cellular and disease ramifications of unrepaired SSB damage.

The Cockayne syndrome B (CSB) protein--defective in a majority of patients suffering from the rare autosomal disorder CS--is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP-DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

Ataxia oculomotor apraxia-1 is a neurological disorder that arises from mutations in the gene encoding the protein aprataxin. A recent study demonstrates that aprataxin is critical for the processing of obstructive DNA termini, suggesting a broader role for DNA single-strand break repair in neurodegenerative disease.

Many cytokines have potential therapeutic applications and several, including type I interferons (IFN), have been approved for the treatment of cancer and/or autoimmune disease. While these agents are effective, they are associated with dose-limiting toxicities that prevent their use at levels sufficient to promote optimal therapeutic benefit. Thus, approaches which enhance the therapeutic index (TI) of cytokines are needed. A moderate degree of tumor-specificity may be achieved by attaching a cytokine such as IFN to a tumor-targeting antibody; such immunocytokines are highly active but show only moderate tumor-specificity since the cytokine is still active on antigen-negative cells. We sought to improve the TI of antibody-targeted cytokines by mutating the cytokine portion to significantly reduce its affinity for its receptor, thereby making it dependent on antibody-targeting. Here we demonstrate that such molecules, consisting of attenuated cytokines (Attenukines™) attached to tumor-targeting antibodies, are 1000–100,000-fold more potent on antigen-positive cells compared to antigen-negative (normal) cells. This is shown for antibody-Attenukine™ fusion proteins based on multiple tumor antigens (CD20, CD38, CD138, HMW-MAA, HLA) and multiple attenuated mutants of IFN, IFN, IL-4 and IL-6. Furthermore, we have evaluated an anti-CD38-attenuated IFN molecule (anti-CD38-Attenukine™) in various CD38+ myeloma xenograft models and found that this molecule retains potent specific anti-tumor efficacy. Moreover, in non-human primates, we have confirmed that the attenuating mutation in IFN indeed decreases non-targeted IFN biomarker responses by greater than 100-fold. Our findings suggest that the administration of antibody-attenukines™ to cancer patients may promote robust cytokine-dependent tumor-killing while minimizing systemic toxicity.

Multiple myeloma (MM) remains an incurable disease despite recent approvals of novel therapies. Treatments which utilize alkylating agents, corticosteroids, proteasome inhibitors and immunomodulatory drugs have provided significant survival benefits; however, tumor relapse eventually occurs with the use of these agents. The cytokine, interferon-α (IFNα), has been used clinically to treat MM for over 30 years. IFNα exerts good anti-myeloma tumor activity but durable responses are rarely achieved due to dose limiting toxicities. Tumor specificity can be modestly increased by attaching IFNα to an anti-CD38 antibody, thus targeting the cytokine to MM cells. However, by introducing an attenuating mutation to the IFNα portion of this fusion protein, the activity of the cytokine is dramatically reduced on CD38 negative cells while exhibiting a similar potency to native IFNα on CD38+ MM cells. As a result, the anti-CD38-attenuated IFNα, referred to as anti-CD38-attenukine™, has over 10,000-fold greater myeloma-specificity than native IFNα in vitro. In vivo, even though the off-target activity is decreased, anti-CD38-attenukine™ has profound anti-tumor effects in murine MM and NHL xenograft models. Treatment with this fusion protein can cure mice with large (>700mm3) tumors—an effect not observed with any other MM agents tested. Here we report that in a Velcade (bortezomib) refractory, IFNα-insensitive primary MM xenograft model, the combination of Velcade with anti-CD38-attenukine™ has potent anti-tumor activity, resulting in tumour elimination and curing of mice. Our findings indicate that anti-CD38-attenukine™ may be a well tolerated, potent anti-MM treatment that may be utilized alone or in combination with Velcade. Citation Format: Sarah Pogue, Tetsuya Taura, Mingying Bi, Glen Mikesell, Yong Yun, Angela Sho, Eric Sanchez, Haiming Chen, James Berenson, Collette Behrens, Maxwell Stevens, Teresa Domagala, Maya Sokolovsky, Hussein Hallak, Moti Rosenstock, Anthony Doyle, David Wilson. Anti-CD38-attenukine: A myeloma-targeting immunocytokine containing an engineered IFNα that provides >10,000-fold enhanced tumor-specific activity compared to native IFNα. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A13.

Sister-chromatid exchange (SCE) is the process whereby, during DNA replication, two sister chromatids break and rejoin with one another, physically exchanging regions of the parental strands in the duplicated chromosomes. This process is considered to be conservative and error-free, since no information is generally altered during reciprocal interchange by homologous recombination. Upon the advent of non-radiolabel detection methods for SCE, such events were used as genetic indicators for potential genotoxins/mutagens in laboratory toxicology tests, since, as we now know, most forms of DNA damage induce chromatid exchange upon replication fork collapse. Much of our present understanding of the mechanisms of SCE stems from studies involving nonhuman vertebrate cell lines that are defective in processes of DNA repair and/or recombination. In this article, we present a historical perspective of studies spearheaded by Dr. Anthony V. Carrano and colleagues focusing on SCE as a genetic outcome, and the role of the single-strand break DNA repair protein XRCC1 in suppressing SCE. A more general overview of the cellular processes and key protein "effectors" that regulate the manifestation of SCE is also presented.

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25 degrees C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25 degrees C in either air (aerobic) or N2 (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25 degrees C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), "Helminthosporium"-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25 degrees C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20-22%. Aflatoxin contamination averaged 174 ng g(-1) over all treatments, and increased up to 798 ng g(-1) in high-moisture grain at stored at 25 degrees C.

Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage. (Cancer Epidemiol Biomarkers Prev 2006;15(5):1000–8)

Repeated marijuana use is known to lead to physical dependence in humans; however, its dependence liability has yet to be adequately assessed in laboratory animals. The goals of the present study were to: assess whether the CB(1) antagonist SR 141716 (rimonabant) precipitates withdrawal in mice that had been repeatedly exposed to marijuana smoke, and to compare these precipitated withdrawal effects to those elicited following intravenous administration of its chief psychoactive component Delta(9)-tetrahydrocannabinol (Delta(9)-THC). SR 141716 elicited a significant increase in paw tremors in mice that were repeatedly dosed with either marijuana or Delta(9)-THC. Unexpectedly, the blood and brain concentrations of Delta(9)-THC following marijuana exposure were considerably lower than those found following Delta(9)-THC injection when comparing an equivalent magnitude of paw tremors in both conditions. Finally, Delta(9)-THC dose-dependently alleviated SR 141716-induced paw tremors in marijuana-dependent mice, but marijuana itself failed to reverse the precipitated withdrawal effect. It is likely that marijuana exposure generated insufficient Delta(9)-THC brain levels (i.e., 203+/-19 ng/g) to reverse the withdrawal signs compared with the brain levels following intravenous injection (i.e., 1862+/-82 ng/g). These findings taken together indicate that mice exposed repeatedly to marijuana smoke exhibit similar precipitated withdrawal effects as Delta(9)-THC-injected mice.

A major role of the multifunctional human Ape1 protein is to incise at apurinic/apyrimidinic (AP) sites in DNA via site-specific endonuclease activity. This nuclease function has been well characterized on double-stranded (ds) DNA substrates, where the complementary strand provides a template for subsequent base excision repair events. Recently, Ape1 was found to incise efficiently at AP sites positioned within the single-stranded (ss) regions of various biologically relevant DNA configurations. The studies within indicated that the ss endonuclease activity of Ape1 is poorly active on ss AP site-containing polyadenine or polythymine oligonucleotides, suggesting a requirement for some form of DNA secondary structure for efficient cleavage. Computational, footprinting, and biochemical analyses indicated that the nature of the secondary structure and the proximity of the AP site influence Ape1 incision efficiency significantly. Replication protein A (RPA), the major ssDNA-binding protein in mammalian cells, was found to bind ss AP-DNA with similar affinity as unmodified ssDNA and ds AP-DNA with lower affinity. Consistent with their known relative DNA binding affinities, RPA blocks/inhibits the ss, but not ds, AP endonuclease function of Ape1. Moreover, RPA inactivates Ape1 incision activity at an AP site within the ss region of a fork duplex, but not a transcription-like bubble intermediate. The data herein suggested a model whereby RPA selectively suppresses the nontemplated ss cleavage activity of Ape1 in vivo, particularly at sites of ongoing replication/recombination, by coating the ssDNA.

1. ABSTRACT The purpose of this paper is to evaluate the post-opera tive Magnetic Resonance Perfusion (MRP) scans of patients undergoing carotid endarterectomy (CEA), using a novel image-analysis algorithm, to determine if post-operative neurocognitive decline is associated with cerebral blood flow changes. CEA procedure reduces the risk of stroke in appropriately selected patients with significant carotid artery stenosis. However, 25% of patients experience subtle cognitive deficits after CEA compared to a control group. It was hypothesized that abnormalities in cerebral blood flow (CBF) are responsible for these cognitive deficits. A novel algorithm for analyzing MRperfusion (MRP) scans to identify and quantify the amount of CBF asymmetry in each hemisphere was developed and to quantify the degree of relative difference between three corresponding vascular regions in the ipsilateral and contralateral hemispheres, the Relative Difference Map (RDM). Patients undergoing CEA and spine surgery (controls) were examined preoperatively, and one day postoperatively with a battery of neuropsychometric (NPM) tests, and labeled “injured” patients with significant cognitive deficits, and “normal” if they demonstrated no decline in neurocognitive function. There are apparently significant RDM differences with MRP scans between the two hemispheres in patients with cognitive deficits which can be used to guide expert reviews of the imagery. The proposed methodology aids in the analysis of MRP parameters in patients with cognitive impairment.

Base excision repair (BER) averts the cytotoxic and mutagenic effects of most endogenously produced DNA damage, including lesions that arise spontaneously due to the intrinsic instability of DNA or modifications that are formed from reactions with intracellular chemicals, such as reactive oxygen species and alkylating agents. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. In its most simplistic form, BER can be fully reconstituted with a minimum of four human proteins and is completed in just five sequential steps: (i) excision of an inappropriate base by a DNA glycosylase (e.g., uracil DNA glycosylase); (ii) incision of the DNA backbone immediately adjacent to the resulting abasic site by apurinic/apyrimidimic endonuclease 1; (iii) removal of the 5′-abasic terminal fragment, and (iv) repair synthesis to fill the gap by DNA polymerase β; and (v) ligation to seal the remaining nick by DNA ligase 1 or a complex of DNA ligase 3 and X-ray repair cross-complementing 1. However, BER can involve the participation of other proteins as well, such as alternative DNA polymerases or one of several nonessential “auxiliary” factors. In addition, BER operates most efficiently when specific protein–protein coordination occurs. Furthermore, several BER protein activities have been shown to be regulated by posttranslational modification, and some of the physical protein interactions link BER to other DNA transaction pathways. In this review, we summarize the current state of the emerging complexities of mammalian BER, focusing on the growing number of reported protein–protein interactions and posttranslational modifications.

Fall armyworm, Spodoptera frugiperda (J.E. Smith), and corn earworm, Helicoverpa zea (Boddie), perennially cause leaf and ear damage to corn, Zea mays L., in the southeastern United States. Transgenic Bacillus thuringiensis (Bt) hybrids with the Bt11, MON810, or 176 events expressing the Cry1Ab insecticidal endotoxin from were evaluated for control fall armyworm and corn earworm at seven locations in Georgia during 1999 and 2000. Corn was planted at the recommended time for each location and 1 and 2 mo later in the southern locations. All Bt events consistently reduced whorl infestation and damage, although event 176 did not prevent whorl damage in the later plantings in the southern locations in both years. All events also reduced seedling damage by the lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in one trial and stalk infestations and tunnel length by southwestern corn borers, Diatraea grandiosella Dyar, in another trial. Hybrids containing Bt11 and MON810 events reduced ear infestations in all trials, although reductions were small in later plantings. Nevertheless, both events reduced grain damage from earworms and armyworms by an average +/- SE of 52.5 +/- 5.1% in all trials. The hybrid containing event 176 did not reduce ear infestations and damage. Total grain aflatoxin concentrations were not significantly affected by Bt resistance in any trial (N = 17). Yield responses were variable with the prevention of yield loss being proportional to the severity of insect damage. Although plantings made after the recommended time did not consistently benefit from Bt resistance, Bt11 and MON810 events were effective in reducing damage to field corn when large infestations occurred. The Bt11 and MON810 events mitigated the risk of severe lepidopteran damage to corn, thereby making later plantings of corn feasible in double-cropping systems.

Werner syndrome patients are deficient in the Werner protein (WRN), which is a multifunctional nuclear protein possessing 3'-5' exonuclease and ATP-dependent helicase activities. Studies of Werner syndrome cells and biochemical studies of WRN suggest that WRN plays a role in several DNA metabolic pathways. WRN interacts with DNA polymerase beta (pol beta) and stimulates pol beta strand displacement synthesis on a base excision repair (BER) intermediate in a helicase-dependent manner. In this report, we examined the effect of the major human apurinic/apyrimidinic endonuclease (APE1) and of pol beta on WRN helicase activity. The results show that WRN alone is able to unwind several single strand break BER intermediates. However, APE1 inhibits WRN helicase activity on these intermediates. This inhibition is likely due to the binding of APE1 to nicked apurinic/apyrimidinic sites, suggesting that APE1 prevents the promiscuous unwinding of BER intermediates. This inhibitory effect was relieved by the presence of pol beta. A model involving the pol beta-mediated hand-off of WRN protein is proposed based on these results.

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level, based on measuring 8-oxoguanine lesions as a biomarker, have led to estimates that vary over three to four orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our findings show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.

Human apurinic/apyrimidinic endonuclease (APE1) is an enzyme of DNA base excision repair (BER) which catalyzes endonucleolytic cleavage immediately 5′ to abasic (AP) sites. APE1 has long been thought to act on AP sites only in double stranded (ds) DNA, in order to generate the appropriate site for insertion of the correct nucleotide of DNA repair synthesis effected by DNA polymerase β. We now present evidence that APE1 also acts on AP sites in single-stranded (ss) DNA. The catalytic efficiency of this activity (defined within as k cat /Km) is ∼20-fold less than the activity against AP sites in ds DNA, with the disparity stemming largely from a difference in Km. Similar to its action on AP sites in ds DNA, catalysis of endonucleolytic cleavage of ss DNA by APE1 is Mg 2+ dependent, DNA N -glycosylase independent, and requires an active site aspartate. In contrast to its activity against AP sites in ds DNA, APE1 does not display product inhibition when acting on an AP site in ss DNA. We suggest that this novel activity is related to the processing of DNA N -glycosylase initiated BER in ss DNA perhaps during replication and/or transcription.

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK(-) osteosarcoma cells that had been depleted of mtDNA (rho(0)) or not (wt). Despite the total absence of mtDNA in the rho(0) cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in rho(0) relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase gamma activities were more affected, 65 and 45% lower, respectively, in rho(0) mitochondria. Overall BER activity in lysates was also about 65% reduced in rho(0) mitochondria. To identify the limiting deficiencies in BER of rho(0) mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase gamma. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase gamma. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in rho(0) mitochondria. While nuclear BER protein levels and activities were generally not altered in rho(0) cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in rho(0) cells, and not a general dysfunction of rho(0) cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.

The kaolin-based particle film Surround has been shown to suppress various insect pests and foliar diseases while reducing canopy temperature and improving water use efficiency in certain agricultural production systems. The usefulness of Surround was examined against important production constraints in peanut, including tomato spotted wilt, leaf spots, and aflatoxin contamination. Field experiments were conducted during 2001 using multi-varietal trials with or without spray treatment of Surround (75 lbs/acre) + NuFilm-17 (8 oz/acre). The effects of Surround application were evaluated for control of thrips and tomato spotted wilt on genotypes Georgia Green, C11-2-39, C34-24, and Sunoleic 97R; for control of leaf spots on AgraTech 201, GK 7 High Oleic, and C-99R; and for control of aflatoxin contamination on Aspergillus-inoculated plots of genotypes Georgia Green, AgraTech 201, and GK 7 High Oleic. In these experiments, Surround applications had little or no effect on thrips populations, tomato spotted wilt severity and incidence, leaf spot severity, drought stress, aflatoxin contamination, chlorophyll content, specific leaf area, and pod yield. In all experiments, host genotype effects were more effective in reducing disease and increasing yield than was Surround protection. Although foliar applications of particle films may be useful for producing certain vegetables and fruits, its benefits for addressing peanut production constraints are limited. Accepted for publication 7 April 2004. Published 19 April 2004.

Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5′ to the damage site. This enzyme also functions in specific contexts to excise 3′-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively “minor” 3′ to 5′ exonuclease activity of Ape1 was found to contribute to the excision of certain 3′-mismatched nucleotides. In this study, I characterize more thoroughly the 3′-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3′-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5′-phosphate, compared to a 5′-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5′-abasic residue exerted an about tenfold attenuation on the 3′ to 5′ exonuclease efficiency of Ape1. A 3′-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase β extension. Ape1 was also found to remove 3′-tyrosyl residues from 3′-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I–DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3′-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3′-flap-type endonuclease activity, again activating these substrates for polymerase β extension.

One approach to the effective treatment of cancer requires the continued development of novel chemotherapeutic agents to kill tumor cells. Additionally, an element of cancer research has been devoted to understanding DNA repair pathways in hopes of defining the factors that confer resistance to anticancer drugs and developing strategies for modulating repair capacity as a means of overcoming resistance or enhancing sensitivity to cancer treatments. Historically, yeast, particularly Saccharomyces cerevisiae, has been used as a model system for DNA repair analyses. Additionally, it has been used to evaluate drug efficacy and selectivity, and to identify new targets for antitumor drugs. The usefulness of yeast for these types of analyses has been primarily because of it being considered to have well-conserved DNA repair processes among eukaryotes. However, as more information has accumulated in mammalian DNA repair, and particularly in DNA base excision repair (BER), a number of striking differences have emerged between yeast and mammalian (human) repair processes. The BER pathway is essential for the repair of damaged DNA induced by oxidizing and alkylating agents, which are the majority of chemotherapeutic drugs used currently in the clinic. The importance of this pathway in processing DNA damage makes its members potential targets for novel chemotherapeutic agents. However, because the BER process and its main players are remarkably divergent from S. cerevisiae to humans, it is worth keeping these differences in mind if yeast continues to be used as a model or primary system in the screening for potential new human therapeutics.

Preharvest aflatoxin contamination (PAC) of peanut occurs under prolonged periods of drought and heat stress. Evaluation of peanut germplasm may identify valuable sources of resistance to PAC, but will require a large scale screening system. The objective of this research was to develop a large-scale field system for screening peanut germplasm for resistance to PAC at Yuma, Arizona. Yuma is located in a desert and has great potential as a site for evaluating germplasm for resistance to PAC. Field studies were conducted in 1990 to determine if aflatoxin contamination would occur in drought stressed peanuts grown at Yuma. Aflatoxin levels up to 2,260 ppb were observed, however, 52% of the plots escaped contamination and the coefficient of variation (C.V.) were unacceptably large. During testing at Yuma in 1990 it was noted that drought stressed plants died quickly due to the rapid exhaustion of soil moisture. A subsurface irrigation system was installed in 1991 to alleviate this problem and allow for an extended period of drought stress in the pod zone. Results for 1991 showed a greatly increased mean aflatoxin contamination, a 50% reduction in the C.V., and a virtual elimination in the occurrence of escapes. A study was conducted in 1992 to compare plots with and without subsurface irrigation to determine if the differences observed between 1990 and 1991 were due to the use of subsurface irrigation. The use of subsurface irrigation in 1992 increased the mean contamination by over 100%, reduced the C.V. by over 50%, and reduced the percentage of escapes by over 90%.

Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts considerably less pathway throughput than observed in experimental in vitro assays. This finding, in combination with the effects of pathway cooperativity on model throughput, supports the hypothesis of cooperation during abasic site repair and between the apurinic/apyrimidinic (AP) endonuclease, Ape1, and the 8-oxoguanine DNA glycosylase, Ogg1. The quantitative model also predicts that for 8-oxoguanine and hydrolytic AP site damage, short-patch Polbeta-mediated BER dominates, with minimal switching to the long-patch subpathway. Sensitivity analysis of the model indicates that the Polbeta-catalyzed reactions have the most control over pathway throughput, although other BER reactions contribute to pathway efficiency as well. The studies within represent a first step in a developing effort to create a predictive model for BER cellular capacity.

The fungal genus Aspergillus was established in 1729, and includes species that are adapted to a wide range of environmental conditions. Many aspergilli produce mycotoxins in foods that may be toxic, mutagenic or carcinogenic in animals. Most of the Aspergillus species are soil fungi or saprophytes but some are capable of causing decay in storage, disease in plants or invasive disease in humans and animals. Major agricultural commodities affected before or after harvest by fungal growth and mycotoxins include corn, peanuts, cottonseed, rice, tree nuts, cereal grains, and fruits. Animal products (meat, milk and eggs) can become contaminated because of diet. Aspergillus flavus, A. parasiticus, A. ochraceus, A. niger, A. fumigatus and other aspergilli produce mycotoxins of concern. These include the aflatoxins and ochratoxins, as well as cyclopiazonic acid, patulin, sterigmatocystin, gliotoxin, citrinin and other potentially toxic metabolites.

Abasic sites and non-conventional 3′-ends, e.g. 3′-oxidized fragments (including 3′-phosphate groups) and 3′-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3′-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3′-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3′-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3′ to 5′ DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered α/β-fold. Lastly, we report that wild-type Ape1 excises 3′-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3′-mismatch repair.

The fungal genus Aspergillus was established in 1729, and includes species that are adapted to a wide range of environmental conditions. Many aspergilli produce mycotoxins in foods that may be toxic, mutagenic or carcinogenic in animals. Most of the Aspergillus species are soil fungi or saprophytes but some are capable of causing decay in storage, disease in plants or invasive disease in humans and animals. Major agricultural commodities affected before or after harvest by fungal growth and mycotoxins include corn, peanuts, cottonseed, rice, tree nuts, cereal grains, and fruits. Animal products (meat, milk and eggs) can become contaminated because of diet. Aspergillus flavus, A. parasiticus, A. ochraceus, A. niger, A. fumigatus and other aspergilli produce mycotoxins of concern. These include the aflatoxins and ochratoxins, as well as cyclopiazonic acid, patulin, sterigmatocystin, gliotoxin, citrinin and other potentially toxic metabolites.

Aim: Renal cell carcinomas (RCCs) is a diverse group of tumors of varying aggressiveness. The development of noninvasive biomarkers of RCC aggressiveness is hindered by a lack of relevant model systems that recapitulate the human situation. In this study, we report the development of patient- derived tumor tissue slice ex vivo and in vivo models in conjunction with clinically translatable non-invasive hyperpolarized (HP) 13C magnetic resonance (MR) imaging technique for the metabolic evaluation of RCC. HP MR is a powerful tool that allows dynamic measurement of specific enzymatic processes implicated in tumorigenesis, and has recently been applied to prostate cancer patients. Methods: Tissue slice cultures (TSCs): RCC tissues were obtained from 8mm cores of nephrectomy cases, precision cut into 300 μm thick disks to allow for maximal oxygen and nutrient diffusion. Long-term tissue viability was assessed by biochemical assays (ATP luciferase, LIVE/DEAD) and phosphorous (31P) MR. Immunohistohcemical staining was also performed to characterize the RCC. Ex vivo evaluation of TSCs in a bioreactor: 4 tissue slices were kept alive in a micro-engineered MR compatible perfusion system. MR data were acquired on a narrow-bore 11.67T Varian INOVA equipped with a 5mm broadband probe. HP 13C MR was acquired dynamically following injection of HP pyruvate and DHA (dehydroascorbate) to assess metabolism in the RCC tissue slices. Animal model: The RCC tissue slices were implanted under the renal capsule of immune-compromised mice. Mice underwent multipramateric proton imaging in addition to dynamic HP 13C MR at 14T (Agilent microimaging system). For the HP MR, a 2D echo-planar based spectral spatial selective pulse sequence was used to obtain dynamic lactate images, post injection of 80mM of HP [1-13C]pyruvate. HP [1-13C] pyruvate has been used to study glycolysis in tumors. Results and Discussion: Ex vivo model: TSCs were viable in the bioreactor for up to 48 hours as confirmed by βNTP levels in 31P spectrum. The 31P spectra displayed characteristic metabolite peaks in the TSCs as seen in RCC patients, with dominant phosphor-ester peaks. Following injection of HP [1-13C] pyruvate into the bioreactor, a dynamic flux to lactate of 0.05 nmols/s/mg tissue was observed in the RCC tissue slices. Additionally we observed the conversion of DHA to Vitamin C in the tumor tissue slices. RCCs are known to have high level of reactive oxygen species, and the combination of this tumor model with HP DHA offers the possibility to interrogate redox potential which is implicated in tumor aggressiveness and treatment response. In vivo model: TSCs were grafted successfully in the sub-renal capsule for even low-grade tumors. Mean tumor ADC measurement of 1.23e-3 mm2/sec in the implanted tumor is similar to that reported in renal cancers clinically. Contrast enhancement of the tumor also confirmed engraftment and perfusion. Elevated HP lactate conversion was observed in the tumor graft, demonstrating the potential of the in vivo model for in-depth metabolic evaluation of tumors that more closely emulate the human situation. IHC of tumor grafts showed the persistence of human vasculature and oncogenic markers. Conclusion: The patient-derive tumor models described above may further enhance our understanding of RCC metabolism and development of clinically relevant biomarkers of tumor aggressiveness and therapy response. Citation Format: Renuka Sriram, Kayvan R. Keshari, Mark Van Criekinge, John Kurhanewicz, David M. Wilson, Donna M. Peehl, Robert Bok, Wang J. Zhen. Patient-derived tissue culture model systems of renal cell carcinoma for development of clinically translatable metabolic biomarkers. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B37.

Base excision repair (BER) is necessary for removal of DNA damage that has occurred from spontaneous decomposition, alkylation and oxidation. Defects in BER have been associated with cancer predisposition, neurodegeneration and premature aging. BER is initiated by a damage-specific DNA glycosylase that excises the damaged base, creating an apurinic/apyrimidinic (AP) site. The AP site is recognized by the multifunctional AP endonuclease 1 (APE1), which cleaves the phosphodiester backbone, producing a DNA strand break. DNA polymerase beta incorporates the correct nucleotide, and BER is completed via ligation by a protein complex of Ligase III and XRCC1. Like many BER proteins, APE1 is essential for survival, as deletion of both alleles in mice leads to embryonic lethality. We are pursuing the hypothesis that more mild reductions in APE1 activity will contribute to disease risk and development. To address this issue, we have engaged two strategies: (1) identification and characterization of APE1 missense mutations and (2) design and characterization of genetically-altered mutant human cell lines. In our first study, we have characterized eight APE1 variants found within the population or identified in endometrial cancer. Our studies reveal that while most of these proteins are normal for protein stability, biochemical activities, and intracellular localization, the endometrial cancer-associated variant Arg237Cys has decreased 3′-functions and AP-DNA binding. In our second study, we have created a heterozygous knockout HCT116 cell line, in which we have deleted one allele of APE1. The heterozygous knockout cells show a loss in AP endonuclease activity and are hypersensitive to DNA damaging drugs. Current cellular strategies are determining the essential nature of APE1 activity in HCT116 and other cell lines Citation Format: Jennifer Illuzzi, Nicole A. Harris, Brittney A. Manvilla, Daemyung Kim, Mengxia Li, Alexander C. Drohat, David M. Wilson. Deciphering the role of APE1 protein variants in disease etiology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2388. doi:10.1158/1538-7445.AM2014-2388

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. APE1 also has 3'-5' DNA exonuclease and 3' DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76-85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3' of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60-80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

The viscous behavior of liquids under nanoconfinement is not well understood. Using a small-amplitude atomic force microscope, we found bulk-like viscosity in a nanoconfined, weakly interacting liquid. A further decrease in viscosity was observed at confinement sizes of a just few molecular layers. Overlaid over the continuum viscous behavior, we measured non-continuum stiffness and damping oscillations. The average stiffness of the confined liquid was found to scale linearly with the size of the confining tip, while the damping scales with the radius of curvature of the tip end.

Several phthalocyanines carrying hydrophobic components have been synthesized and shown to bind to a group of cyclodextrin dimers with a carbon−carbon double bond in the linker. The complexes are soluble in water. On irradiation in the presence of oxygen, the singlet oxygen produced cleaves the olefinic linkers in the complexes, resulting in precipitation of the sensitizers. This process concentrates the sensitizers in the light beam, a process that has useful potential in photodynamic therapy.

International audience; The human ISG20/HEM45 gene was identified independently on the basis of its increased level of expression in response to either interferon or estrogen hormone. Notably, the encoded protein is homologous with members of the 3' to 5' exonuclease superfamily that includes RNases T and D, and the proofreading domain of Escherichia coli DNA polymerase I. We provide here direct biochemical evidence that Isg20 acts as a 3' to 5' exonuclease in vitro. This protein displays a pH optimum of approximately 7.0, prefers Mn2+ as a metal cofactor, and degrades RNA at a rate that is approximately 35-fold higher than its rate for single-stranded DNA. Along with RNase L, Isg20 is the second known RNase regulated by interferon. Previous data showed that Isg20 is located in promyelocytic leukemia (PML) nuclear bodies, known sites of hormone-dependent RNA polymerase II transcription and oncogenic DNA viral transcription and replication. The combined data suggest a potential role for Isg20 in degrading viral RNAs as part of the interferon-regulated antiviral response and/or cellular mRNAs as a regulatory component of interferon and estrogen signaling.

Phenotypic expression of primary hyperoxaluria: Comparative features of types I and II. Background The primary hyperoxalurias are autosomal recessive disorders resulting from deficiency of hepatic alanine:glyoxylate aminotransferase (PHI) or D-glycerate dehydrogenase/glyoxylate reductase (PHII). Marked hyperoxaluria results in urolithiasis, renal failure, and systemic oxalosis. A direct comparison of PHI and PHII has not previously been available. Methods Twelve patients with PHI and eight patients with PHII with an initial creatinine clearance of greater than or equal to 50 mL/min/1.73 m 2 underwent similar laboratory evaluation, clinical management, and follow-up. Diagnosis of PHI and PHII was made by hepatic enzyme analysis ( N = 11), increased urinary excretion of glycolate or glycerate ( N = 7), or complete pyridoxine responsiveness ( N = 2). Six PHI and five PHII patients had measurements of calcium oxalate crystalluria, urine supersaturation, and urine inhibition of calcium oxalate crystal formation. Results PHI and PHII did not differ in age at the onset of symptoms, initial serum creatinine, or plasma oxalate concentration. Urine oxalate excretion rates were higher in PHI (2.19 ± 0.61 mmol/1.73 m 2 /24 hours) than PHII (1.61 ± 0.43, P = 0.04). Urine osmolality, calcium, citrate, and magnesium concentrations were lower in PHI than PHII ( P = 0.001, P = 0.019, P = 0.0002, P = 0.03, respectively). Crystalluria scores and calcium oxalate inhibitory activity of the urine did not differ between PHI and PHII. Calcium oxalate supersaturation in the urine was less in PHI (7.3 ± 1.9) compared with PHII (14.0 ± 3.3, P = 0.002). During follow-up of 10.3 ± 9.6 years in PHI and 18.1 ± 5.6 years in PHII, stone-forming activity and stone procedures were more frequent in PHI than PHII ( P P = 0.01, respectively). Four of 12 PHI compared with 0 of 8 PHII patients progressed to end-stage renal disease ( P = 0.03). Conclusion The severity of disease expression is greater in type I primary hyperoxaluria than in type II. The difference may be due to greater oxalate excretion and lower concentrations of urine citrate and magnesium in patients with PHI compared with PHII.

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5' to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited approximately 40-60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit approximately 10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.

The reemergence on the debate of the use of marijuana for medicinal purposes has been the impetus for developing an acceptable delivery form of aerosolized cannabinoids. The goals of the present study were to: (1) develop and characterize the physical properties of an aerosolized form of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive constituent present in marijuana; and (2) assess the pharmacological effects of cannabinoid inhalation in mice. A Small Particle Aerosol Generator (SPAG) nebulizer, used to generate the aerosol, had an output of approximately 0.154 mg/l of aerosolized Delta(9)-THC with a 2.0 microm mass median aerodynamic diameter and a 2.2 geometric standard deviation (GSD). Virtually all the particles were less than 5.0 microm in diameter suggesting that they were sufficiently small to penetrate deeply into the lungs. Inhalation exposure to aerosolized Delta(9)-THC in mice elicited antinociceptive effects that were dependent on concentration and exposure time with an estimated Delta(9)-THC dose of 1.8 mg/kg. On the other hand, inhalation exposure to Delta(9)-THC failed to produce two other indices indicative of cannabinoid activity, hypothermia and decreases in spontaneous locomotor activity. The antinociceptive effects occurred within 5 min of exposure and lasted approximately 40 min in duration. The cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR 141716A), but not naloxone, blocked these antinociceptive effects (AD(50)=0.09 mg/kg) indicating a cannabinoid receptor mechanism of action. Similarly, inhalation exposure to a water soluble cannabinoid analog, 3-(5'-cyano-1', 1'dimethylheptyl)-1-(4-N-morpholinobutyrloxy)-Delta(8)-te trahydrocann abinol (O-1057), produced antinociception that was blocked by SR 141716A. These results demonstrate that the development of an aerosolized form of cannabinoids for human medicinal use is feasible.

We report here the identification of human homologues to the essential Escherichia coli Orn protein and the related yeast mitochondrial DNA-escape pathway regulatory factor Ynt20. The human proteins appear to arise from alternatively spliced transcripts, and are thus identical, except the human Ynt20 equivalent contains an NH(2)-terminal extension that possesses a predicted mitochondrial protease cleavage signal. In vitro analysis revealed that the smaller human protein exhibits a 3' to 5' exonuclease activity for small (primarily/=5 nucleotides in length) single-stranded RNA and DNA oligomers. We have named this human protein Sfn for small fragment nuclease to reflect its broad substrate range, and have termed the longer protein hSfnalpha. Sfn prefers Mn(2+) as a metal cofactor and displays a temperature-resistant (to 50 degrees C) nuclease activity. Kinetic analysis indicates that Sfn exhibits a similar affinity for small RNAs and DNAs (K(m) of approximately 1.5 micrometer), but degrades small RNAs approximately 4-fold more efficiently than DNA. Mutation of a conserved aspartate (Asp(136)) to alanine abolishes both nuclease activities of Sfn. Northern blot analysis revealed that a 1-kilobase transcript corresponding to SFN and/or SFNalpha (these mRNAs differ by only two nucleotides) is expressed at varying levels in all fetal and adult human tissues examined. Expressed tag sequence clone analysis found that the two splice variants, SFN to SFNalpha, are present at a ratio of roughly 4 to 1, respectively. The results presented within suggest a role for human Sfn in cellular nucleotide recycling.

Our understanding of early human development has been impeded by the general difficulty in obtaining suit-able samples for study. As a result, and because of the extraordinarily high degree of evolutionary conserva-tion of many developmentally important genes and developmental pathways, great reliance has been placedon extrapolation from animal models of development, principally the mouse. However, the strong evolution-ary conservation of coding sequence for developmentally important genes does not necessarily mean thattheir expression patterns are as highly conserved. The very recent availability of human embryonic samplesfor gene expression studies has now permitted for the first time an assessment of the degree to which we canconfidently extrapolate from studies of rodent gene expression patterns. We have found significant human–mouse differences in embryonic expression patterns for a variety of genes. We present detailed data for twoillustrative examples. Wnt7a, a very highly conserved gene known to be important in early development,shows significant differences in spatial and temporal expression patterns in the developing brain (midbrain,telencephalon) of man and mice. CAPN3, the locus for LGMD2A limb girdle muscular dystrophy, and itsmouse orthologue differ extensively in expression in embryonic heart, lens and smooth muscle. Our studyalso shows how molecular analyses, while providing explanations for the observed differences, can be impor-tant in providing insights into mammalian evolution.INTRODUCTIONAlthough the structural genomics phase of the human genomeproject is nearing completion, and will shortly provide a completeinventory of human genes, our knowledge of human gene expres-sion is rudimentary (1). High resolution gene expression studiesare most conveniently carried out on the miniaturized tissues ofthe embryo, but the general difficulty in accessing suitable mate-rial for studying early human development and the widespreadbelief that early mammalian development is essentially conservedhave prompted widespread studies of rodent models, notably themouse. The resulting reliance on rodent embryonic expressionstudies makes it important to know just how far we can extrapo-late from them with confidence.The huge emphasis on gene expression in rodent embryos hasbeen justified by strong similarities in the organization andmorphology of vertebrate embryos and by the extraordinaryevolutionary conservation of many developmental pathways andof key genes that are known or expected to be important in regu-lating early development. Species differences in development canbe represented by an hourglass model (2,3): very considerabledifferences exist at very early stages in development, such asgastrulation and neurulation, and morphological divergence canbe extensive in later development,but at intermediate stages whenthe body plan is being laid down embryonic development hasbeen widely considered to be highly conserved during vertebrateevolution. However, the idea that there is a conserved embryonic(phylotypic) stage in vertebrates has not been supported bydetailed comparative studies: stage-matched embryos fromclosely related vertebrate and mammalian species were recentlyshown to exhibit considerable morphological differences (4).Recent molecular studies have also emphasized unexpectedspecies differences and raised concerns regarding the extent to

Genetic recombination involves either the homo-logous exchange of nearly identical chromosome regions or the direct alignment, annealing and ligation of processed DNA ends. These mechanisms are involved in repairing potentially lethal or mutagenic DNA damage and generating genetic diversity within the meiotic cell population and antibody repertoire. We report here the identification of a mouse gene, termed mExo1 for mouse exonuclease 1, which encodes a approximately 92 kDa protein that shares homology to proteins of the RAD2 nuclease family, most notably human 5' to 3' exonuclease Hex1/hExo1, yeast exonuclease 1 (Exo1) proteins and Drosophila melanogaster Tosca. The mExo1 gene maps to distal chromosome 1, consistent with the recent mapping of the orthologous HEX1 / hEXO1 gene to chromosome 1q42-q43. mExo1 is expressed prominently in testis, an area of active homologous recombination, and spleen, a prominent lymphoid tissue. An increased level of mExo1 mRNA was observed during a stage of testis development where cells that are actively involved in meiotic recombination arise first and represent a significant proportion of the germ cell population. Comparative evaluation of the expression patterns of the human and mouse genes, combined with previous biochemical and yeast genetic studies, indicate that the Exo1-like proteins are important contributors to chromosome processing during mammalian DNA repair and recombination.

Ape1, the major protein responsible for excising apurinic/apyrimidinic (AP) sites from DNA, cleaves 5' to natural AP sites via a hydrolytic reaction involving Mg2+. We report here that while Ape1 incision of the AP site analog tetrahydrofuran (F-DNA) was approximately 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and propane (P-DNA) acyclic abasic site analogs was only 20 and 30-fold lower, respectively, in EDTA compared to Mg2+. This finding suggests that the primary role of the metal ion is to promote a conformational change in the ring-containing abasic DNA, priming it for enzyme-mediated hydrolysis. Mutating the proposed metal-coordinating residue E96 to A or Q resulted in a approximately 600-fold reduced incision activity for both P and F-DNA in Mg2+compared to wild-type. These mutants, while retaining full binding activity for acyclic P-DNA, were unable to incise this substrate in EDTA, pointing to an alternative or an additional function for E96 besides Mg2+-coordination. Other residues proposed to be involved in metal coordination were mutated (D70A, D70R, D308A and D308S), but displayed a relatively minor loss of incision activity for F and P-DNA in Mg2+, indicating a non-essential function for these amino acid residues. Mutations at Y171 resulted in a 5000-fold reduced incision activity. A Y171H mutant was fourfold less active than a Y171F mutant, providing evidence that Y171 does not operate as the proton donor in catalysis and that the additional role of E96 may be in establishing the appropriate active site environment via a hydrogen-bonding network involving Y171. D210A and D210N mutant proteins exhibited a approximately 25,000-fold reduced incision activity, indicating a critical role for this residue in the catalytic reaction. A D210H mutant was 15 to 20-fold more active than the mutants D210A or D210N, establishing that D210 likely operates as the leaving group proton donor.