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Propylene glycol (PG), a simple alcohol, is a commonly used vehicle for aerosol dosage formulations. Quantification of PG in plasma and lung tissue is, therefore, important for new drug development. We describe a highly sensitive and selective method for the quantitative determination of PG in rat plasma and lung tissue, using liquid chromatography (LC) with positive atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS) detection. Propylene glycol and the internal standard (IS) 1,4‐butanediol were derivatized with benzoyl chloride under alkaline conditions to enhance the sensitivity of detection. Ionization efficiency was improved following derivatization. The limits of detection (LOD) for rat plasma and rat lung tissue were 0.269 µg/mL and 1.12 µg/g, respectively. The LOQs for rat plasma and lung tissue were 0.448 µg/mL and 1.62 µg/g, respectively. Calibration curves were linear from 2 to 4000 µg/mL for rat plasma (r = 0.9990) and from 2 to 2400 µg/g for rat lung tissue (...

Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism. Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities. Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability. However, strains lacking Rad27 are viable. The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication. We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency. While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells. This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER). Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.

Several resistance sources and resistance mechanisms to aflatoxin formation and corn earworm (Helicoverpazea Boddie) damage to maize (Zeamays L.) have been identified. Based on this knowledge, experiments were initiated toward achievement of the following objectives: (1) to confirm earlier determinations on resistance traits of germplasm sources and to identify quantitative trait loci (QTL) associated with each of the traits, and (2) upon estimation of the degree of QTL effects on each trait, to generate a maize population, with chemical and physical resistance to Aspergillus spp. and ear-feeding insects, for inbred development. A 2-year field experiment to evaluate selected genotypes inoculated with A. flavus and infested with corn earworm revealed that significant variation exists among the genotypes for aflatoxin contamination and corn earworm damage. The protection of maize ears against aflatoxin contamination was primarily dependent on resistance to fungal infection and ear-feeding insects, and excellent husk coverage and tightness. A major QTL (p1) identified on chromosome 1S had effects of 54.0, 42.1, and 28.3% on the phenotypic variability for concentrations of silk maysin, 3′-methoxymaysin+apimaysin, and chlorogenic acid, respectively. Markers/QTLs for husk phenotypic traits and total aflatoxin concentrations have been determined, but more detailed mapping of these chromosomic regions will be necessary to locate precise markers/QTLs for husk traits and aflatoxin production. Realizing the complexity of the Aspergillus–aflatoxin-maize system and the factors affecting aflatoxin contamination, we are directing our program toward marker-assisted breeding to enhance or improve general genetic resistance to ear-feeding insects and invasion by Aspergillus spp., A. Butron thanks ‘Ministerio de Educacion y Ciencia’ of Spain that supported her while she was carrying out the present study. Research was supported by funds provided by USDA Agricultural Research Service and by the Georgia Agricultural Commodity Commission for Corn., MEC, USDA Agricultural Research Service, Georgia Agricultural Commodity Commission for Corn

beta-cyclodextrin-based receptors were synthesized and tested for their ability to induce a helical fold in peptides bearing hydrophobic amino acid residues in the i, i+11- or i, i+14-positions. Circular dichroism experiments revealed that a dimeric beta-cylodextrin receptor synthesized from a [1,1'-biphenyl]-4,4'-dithiol core demonstrated an ability to fold a designed peptide bearing the artificial amino acid L-p-t-butylphenylalanine in the i, i+11-positions, while other dimeric and monomeric receptors failed to do so. Titration studies were performed using both circular dichroism and calorimetry, the analysis of which yielded an apparent K(a) on the order of 10(4)-10(5) M(-1). However, no evidence could be obtained for helical folding with a peptide carrying tryptophan residues in place of the p-t-butylphenylalanine units. Our studies suggest that receptors of this type may be useful in molecular recognition of hydrophobic, already alpha-helical peptides in aqueous solution.

Hydrogen bonded histidine-aspartate (His-Asp) pairs are critical constituents in several key enzymatic reactions. To date, the role that these pairs play in catalysis is best understood in serine and trypsin-like proteases, where structural and biochemical NMR studies have revealed important pK(a) values and hydrogen bonding patterns within the catalytic pocket. However, the role of the His-Asp pair in metal-assisted catalysis is less clear. Here, we apply liquid-state NMR to investigate the role of a critical histidine residue of apurinic endonuclease 1 (Ape1), a human DNA repair enzyme that cleaves adjacent to abasic sites in DNA using one or more divalent cations and an active-site His-Asp pair. The results of these studies suggest that the Ape1 His-Asp pair does not function as either a general base catalyst or a metal ligand. Rather, the pair likely stabilizes the pentavalent transition state necessary for phospho-transfer.

Proteinuria is an important marker of kidney disease. Simple methods to determine the presence of proteinuria in a semiquantitative fashion require measurement of either a protein-creatinine or protein-osmolality ratio.Urine samples from 134 healthy infants and children and 150 children from the pediatric nephrology practice were analyzed to develop normative data for protein-osmolality ratios on random urine samples and compare protein-osmolality with protein-creatinine ratio as a predictor of 24-hour urine protein excretion. Children were grouped according to age. Three groups were established: infants (2 years), younger children (2 to 8 years), and older children (9 to 18 years). An adult cohort was similarly analyzed.For healthy children older than 2 years, the optimal value discriminating normal from abnormal protein excretion was determined to be a protein-osmolality ratio of 0.15 mg x kg H2O/mOsm. L; for children between 2 and 8 years old, 0.14; and for children older than 8 years, 0.17 (P = not significant between age groups). The corresponding optimal cutoff value for protein-creatinine ratio for the entire group of children older than 2 years is 0.20. Area under the curve analysis of receiver operator characteristic curves showed protein-creatinine ratio was superior to protein-osmolality ratio for predicting abnormal amounts of proteinuria in children and adolescents (P0.0001). In adults, both ratios are equally accurate.Given the superiority of protein-creatinine ratio in children, it would be appropriate to screen urine samples for proteinuria using protein-creatinine ratio rather than protein-osmolality ratio.

HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5' to 3' exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from -6240 to +1600bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning -688 to +1600bp that exhibits full transcriptional activity while a slightly shorter fragment from -658 to +1600bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with -687 to -681bp and -665 to -658bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the -687 to -681bp element, while transcription factors Sp1 and Sp3 recognize the -665 to -658bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.

The progress and problems in the Caribbean Amblyomma Program (CAP) are reviewed since its inception in 1995. During 1998, there were funding and administrative management problems. USDA resolved the acute funding crisis, and after three years of negotiation, the CAP has now secured an additional euro 1.5 million from the European Community. Changes in administration in 1998 included the withdrawal of IICA from the program, and the transition during the decentralization of administrative and financial management from FAO headquarters to the Regional Office for Latin America and the Caribbean, based in Chile. A general overview of technical progress and one case study, St. Kitts, is presented. One major concern that emerged during 2000 is that the elimination of the small remaining tropical bont tick (TBT) “hot spots” in both St. Kitts and St. Lucia remained elusive. Why is this so? Egrets? Alternative residual hosts? Or is it fatigue in both technical and administrative management functions? Of even greater concern is the finding of two, apparently new, foci in St. Croix (USVI) in the north and St. Vincent in the south. A critical overview of the program has identified one major remaining constraint—an appropriate management support function at both regional and, in some countries, at the national level. A proposal for a revised management strategy, coupled with the identification of a future strategy to succeed the CAP, namely a Caribbean Animal Resources Management (CARM) Program.

The goal of the present study was to formulate a Delta(9)-tetrahydrocannabinol (Delta(9)-THC) metered-dose inhaler (MDI) that can be used to provide a systemic dose of Delta(9)-THC via inhalation. Following physiochemical characterization and accelerated stability testing of the aerosol, mice were exposed to the aerosol and evaluated for pharmacological effects indicative of cannabinoid activity, including hypomotilìty, antinociception, catalepsy, and hypothermia. The fine particle dose of Delta(9)-THC was 0.22 +/- 0.03 mg (mean +/- S.D.) or 25% of the emitted dose and was not affected by accelerated stability testing. A 10-min exposure to aerosolized Delta(9)-THC elicited hypomotility, antinociception, catalepsy, and hypothermia. Additionally, Delta(9)-THC concentrations in blood and brain at the antinociceptive ED(50) dose were similar for both inhalation and intravenous routes of administration. Finally, pretreatment with the CB(1) receptor antagonist SR 141716A (10 mg/kg, i.p.) significantly antagonized all of the Delta(9)-THC-induced effects. These results indicate that an MDI is a viable method to deliver a systemic dose of Delta(9)-THC that elicits a full spectrum of cannabinoid pharmacological effects in mice that is mediated via a CB(1) receptor mechanism of action. Further development of a Delta(9)-THC MDI could provide an appropriate delivery device for the therapeutic use of cannabinoids, thereby reducing the need for medicinal marijuana.

Human Exo1 is a member of the RAD2 nuclease family with roles in replication, repair and recombination. Despite sharing significant amino acid sequence homology, the RAD2 proteins exhibit disparate nuclease properties and biological functions. In order to identify elements that dictate substrate selectivity within the RAD2 family, we sought to identify residues key to Exo1 nuclease activity and to characterize the molecular details of the human Exo1-DNA interaction. Site-specific mutagenesis studies demonstrate that amino acids D78, D173 and D225 are critical for Exo1 nuclease function. In addition, we show that the chemical nature of the 5'-terminus has a major impact on Exo1 nuclease efficiency, with a 5'-phosphate group stimulating degradation 10-fold and a 5'-biotin inhibiting degradation 10-fold (relative to a 5'-hydroxyl moiety). An abasic lesion located within a substrate DNA strand impedes Exo1 nucleolytic degradation, and a 5'-terminal abasic residue reduces nuclease efficiency 2-fold. Hydroxyl radical footprinting indicates that Exo1 binds predominantly along the minor groove of flap DNA, downstream of the junction. As will be discussed, our results favor the notion that the single-stranded DNA structure is pinched by the helical arch of the protein and not threaded through this key recognition loop. Furthermore, our studies indicate that significant, presumably biologically relevant, differences exist between the active site dynamics of Exo1 and Fen1.

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) 13 C dehydroascorbate (DHA) and 13 C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7 days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP 13 C vitamin C/(vitamin C + DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP 13 C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP 13 C lactate/pyruvate ratio at day 7 compared with baseline, although the 13 C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham-operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP 13 C MRS for the non-invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.

Etheno (ε) DNA base adducts are highly mutagenic lesions produced endogenously via reactions with lipid peroxidation (LPO) products. Cancer-promoting conditions, such as inflammation, can induce persistent oxidative stress and increased LPO, resulting in the accumulation of ε-adducts in different tissues. Using a recently described fluorescence multiplexed host cell reactivation assay, we show that a plasmid reporter bearing a site-specific 3,N4-ethenocytosine (εC) causes transcriptional blockage. Notably, this blockage is exacerbated in Cockayne Syndrome and xeroderma pigmentosum patient-derived lymphoblastoid and fibroblast cells. Parallel RNA-Seq expression analysis of the plasmid reporter identifies novel transcriptional mutagenesis properties of εC. Our studies reveal that beyond the known pathways, such as base excision repair, the process of transcription-coupled nucleotide excision repair plays a role in the removal of εC from the genome, and thus in the protection of cells and tissues from collateral damage induced by inflammatory responses.

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) (13)C MR and interrogated HP [1-(13)C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP (13)C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra- (Lacin ) and extracellular (Lacex ) HP lactate signals were robustly resolved in dynamic (13)C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1-(13)C]pyruvate delivery, the ratio of HP Lacin /Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p0.05) increase in the Lacex pool size. Lacin /Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient-derived RCC tissue slices using HP [1,2-(13)C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-(13)C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export.

Oxidative stress has been proposed to be a unifying cause for diabetic nephropathy and a target for novel therapies. Here we apply a new endogenous reduction-oxidation (redox) sensor, hyperpolarized (HP) 13C dehydroascorbate (DHA), in conjunction with MRI to noninvasively interrogate the renal redox capacity in a mouse diabetes model. The diabetic mice demonstrate an early decrease in renal redox capacity, as shown by the lower in vivo HP 13C DHA reduction to the antioxidant vitamin C (VitC), prior to histological evidence of nephropathy. This correlates with lower tissue reduced glutathione (GSH) concentration and higher NADPH oxidase 4 (Nox4) expression, consistent with increased superoxide generation and oxidative stress. ACE inhibition restores the HP 13C DHA reduction to VitC with concomitant normalization of GSH concentration and Nox4 expression in diabetic mice. HP 13C DHA enables rapid in vivo assessment of altered redox capacity in diabetic renal injury and after successful treatment.

The apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox-dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein-protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence-based high-throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas.

Temperature can modify neural and behavioral responses to taste stimuli that elicit “sweetness,” a perception linked to intake of calorie-laden foods. However, the role of temperature in the neural representation of sweet taste is poorly understood. Here we made electrophysiological recordings from gustatory neurons in the medulla of inbred mice to study how adjustments in taste solution temperature to cool (18°C), ambient (22°C), and warm (30°C and 37°C) values changed the magnitude and latency of gustatory activity to sucrose (0, 0.05, 0.1, 0.17, 0.31, and 0.56 M). Analysis of 22 sucrose-best neurons revealed that temperature markedly influenced responses to sucrose, which, across concentrations, were largest when solutions were warmed to 30°C. However, reducing solution temperature from warm to ambient to cool progressively steepened the slope of the sucrose concentration-response function computed across cells ( P < 0.05), indicating that mean activity to sucrose increased more rapidly with concentration steps under cooling than with warming. Thus the slope of the sucrose concentration-response function shows an inverse relation with temperature. Temperature also influenced latency to the first spike of the sucrose response. Across neurons, latencies were shorter when sucrose solutions were warmed and longer, by hundreds of milliseconds, when solutions were cooled ( P < 0.05), indicating that temperature is also a temporal parameter of sucrose activity. Our findings reveal that temperature systematically modifies the timing of gustatory activity to sucrose in the mammalian brain and how this activity changes with concentration. Results further highlight how oral somatosensory cues function as physiological modulators of gustatory processing.

Objective—To assess perioperative findings and postoperative complications and outcomes in dogs that had ectopic thyroid carcinomas with invasion into the hyoid apparatus and underwent tumor excision with partial hyoidectomy. Design—Retrospective case series. Animals—5 client-owned dogs. Procedures—Medical records of dogs that had an ectopic neuroendocrine tumor with invasion into the hyoid apparatus and underwent tumor excision with partial hyoidectomy were reviewed for information regarding perioperative and postoperative findings and outcome. During surgery in each case, the thyrohyoid and ceratohyoid or epihyoid bones (depending on degree of hyoid apparatus involvement) were sharply transected, allowing en bloc removal of the tumor. The ipsilateral cut ends of the thyrohyoid and ceratohyoid or epihyoid bones (depending on which was cut) were sutured together with polypropylene suture in a simple interrupted pattern. Results—All partial hyoidectomy procedures were completed without surgical or anesthetic complications. All 5 dogs were able to eat and drink between 7 and 24 hours after surgery, with no signs of dysphagia, ptyalism, or abnormal tongue carriage. Follow-up information was obtained over a period of 173 to 587 days after surgery for all 5 dogs; 4 dogs were still alive at last follow-up. One dog was euthanized 587 days after surgery because of lethargy, inappetence, and hypercalcemia. Conclusions and Clinical Relevance—From this limited series of cases, results suggested that partial resection of the hyoid apparatus during removal of ectopic thyroid carcinoma may be tolerated well and be associated with very good functional outcomes in dogs. (J Am Vet Med Assoc 2014;245:1319–1324)

The study of transient chemical phenomena by conventional NMR has proved elusive, particularly for non-1H nuclei. For 13C, hyperpolarization using the dynamic nuclear polarization (DNP) technique has emerged as a powerful means to improve SNR. The recent development of rapid dissolution DNP methods has facilitated previously impossible in vitro and in vivo study of small molecules. This review presents the basics of the DNP technique, identification of appropriate DNP substrates, and approaches to increase hyperpolarized signal lifetimes. Also addressed are the biochemical events to which DNP-NMR has been applied, with descriptions of several probes that have met with in vivo success.

FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p = 4.89 × 10(-57)), high mitotic index (p = 5.25 × 10(-28)), pleomorphism (p = 6.31 × 10(-19)), ER negative (p = 9.02 × 10(-35)), PR negative (p = 9.24 × 10(-24)), triple negative phenotype (p = 6.67 × 10(-21)), PAM50.Her2 (p = 5.19 × 10(-13)), PAM50. Basal (p = 2.7 × 10(-41)), PAM50.LumB (p = 1.56 × 10(-26)), integrative molecular cluster 1 (intClust.1) (p = 7.47 × 10(-12)), intClust.5 (p = 4.05 × 10(-12)) and intClust. 10 (p = 7.59 × 10(-38)) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p = 4.4 × 10(-16)) and multivariate analysis (p = 9.19 × 10(-7)). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps < 0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps < 0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps < 0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps < 0.05). We conclude that FEN1 is a promising biomarker in breast and ovarian epithelial cancer.

Objectives (1) Describe arthroscopic BURP surgical technique, (2) assess association of visual control and surgeon experience to tenotomy completeness and regional iatrogenic tissue damage. Study Design Cadaveric study. Sample Population Canine cadavers weighing >20 kg (n = 16; 32 elbows). Methods Phase 1 = dissection/anatomic description/procedural refinement (n = 6). Phase 2 = technique description (n = 6). Phase 3 = association of surgeon experience and procedural visual control to tenotomy completion and regional iatrogenic damage (n = 20). Elbows were randomly assigned via coin toss to an experienced- or inexperienced-arthroscopist. Using conventional medial portals, surgeons sought to identify the medial collateral ligament (MCL) and ulnar insertion of the biceps tendon (uBT) before attempting complete tenotomy. Upon procedural completion, surgeons assigned a standardized “visual control score” (VCS) describing viewing that governed procedure and predicted % uBT release, MCL, and median nerve damage. Post-procedural dissection determined actual tenotomy completion and iatrogenic tissue damage. Results Complete BURP was achieved in 16 of 19 elbows. VCS was associated with tenotomy completeness (P

A family of oxide fibers, Nextel™ 610 Ceramic Oxide Fiber, Nextel™ 720 Ceramic Oxide Fiber and a new fiber, Nextel™ 650 Ceramic Oxide Fiber, has been developed specifically for the reinforcement of metal and ceramic matrix composites. This paper summarizes room and high temperature properties for these fibers. The strength of both single filaments and multi-filament rovings of Nextel 610, 650 and 720 fibers was determined between 25 and 250 mm gauge length. Weibull analysis was used to compare the statistical fracture distribution and gauge length dependence of strength. Fiber fracture statistics were in accord with Weibull theory; the effect of diameter variability on the statistical analysis was found to be small. Fractographic analysis on Nextel 610 fiber was used to identify primary fracture-causing defects; defect size was correlated with Griffith fracture predictions. High temperature single filament strength measurements were performed on Nextel 610, 650 and 720 fibers between 800 and 1400°C. High temperature strength varied inversely with strain rate. In combination with tensile creep tests at 1100 and 1200°C, these were used to compare the elevated temperature capability of each fiber and determine maximum use temperatures. The development of crystalline yttrium aluminum garnet fibers that demonstrate further improvements in creep performance relative to Nextel 720 fibers is also discussed.

Although the majority of cannabinoid users smoke marijuana, the preponderance of laboratory animal research is based on administration of Delta9-tetrahydrocannabinol (Delta9-THC) or other cannabinoid agents via injection. The aim of the present study was to evaluate the impact of inhaling marijuana, or ethanol-extracted placebo smoke in the mouse model of cannabinoid activity by assessing inhibition of spontaneous activity, antinociception, catalepsy, and body temperature. In order to determine dosimetry, blood levels of Delta9-THC were obtained following either marijuana exposure or intravenous injection of Delta(9)-THC. Inhalation exposure to marijuana produced dose-related increases in antinociception and catalepsy, with estimated ED50 doses of Delta9-THC of 2.4 and 3.8 mg/kg, respectively. However, hypothermia and locomotor depression occurred in both the placebo- and marijuana-exposed mice. The CB1 receptor antagonist, SR 141716A antagonized the antinociceptive effects of marijuana (AD50 = 0.6 mg/kg), but only slightly decreased marijuana-induced catalepsy, and failed to alter either the hypothermic or locomotor depressive effects. In contrast, SR 141716A antagonized the antinociceptive, cataleptic, and hypothermic effects of intravenously administered Delta9-THC in mice that were exposed to air alone, though all subjects exhibited locomotor depression, possibly related to the restraint. In accordance with reports of others, these data suggest that exposure to smoke alone has pharmacological consequences. Our findings also indicate that marijuana-induced antinociception is mediated through a CB1-receptor mechanism of action and are consistent with the notion that Delta9-THC is mainly responsible for this effect.

The results obtained from two consecutive functional analyses conducted with a 6-year-old child with autism are described. In the initial functional analysis, the highest rates of problem behavior occurred in the play condition. In that condition, the delivery of attention appeared to occasion problem behaviors. A second functional analysis was conducted wherein an escape from attention condition and a tangible condition were added. In the second functional analysis, higher rates of responding were observed in the escape from attention and tangible conditions. The results suggested that problem behavior was maintained by negative reinforcement in the form of escape from attention and positive reinforcement in the form of gaining access to preferred tangible items. Problem behavior was treated using functional communication training combined with noncontingent reinforcement.

DNA continuously suffers the loss of its constituent bases, and thereby, a loss of potentially vital genetic information. Sites of missing bases--termed abasic or apurinic/apyrimidinic (AP) sites--form spontaneously, through damage-induced hydrolytic base release, or by enzyme-catalyzed removal of modified or mismatched bases during base excision repair (BER). In this review, we discuss the structural and biological consequences of abasic lesions in DNA, as well as the multiple repair pathways for such damage, while emphasizing the mechanistic operation of the multi-functional human abasic endonuclease APE1 (or REF-1) and its potential relationship to disease.

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3′ replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca 2+ exhibits complex stimulatory and inhibitory effects on the Mg 2+ -dependent catalysis of Ape1, even though Ca 2+ itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

Peanuts become contaminated with aflatoxins when subjected to prolonged periods of heat and drought stress. The effect of drought tolerance on aflatoxin contamination is not known. The objectives of this research were to evaluate preharvest aflatoxin contamination in peanut genotypes known to have drought tolerance and to determine the correlation of drought tolerance characteristics with aflatoxin contamination. Twenty genotypes with different levels of drought tolerance were grown in Yuma, AZ (a desert environment) and under rain-protected shelters in Tifton, GA. Two drought-tolerant genotypes (PI 145681 and Tifton 8) and an intolerant genotype (PI 196754) were selected for further examination in a second experiment with two planting dates in 1997 at Tifton. Drought and heat stress conditions were imposed for the 40 d preceding harvest. The drought-intolerant genotype had greater preharvest aflatoxin contamination than Florunner (the check cultivar) in the tests conducted in 1997. Both drought-tolerant genotypes had less preharvest aflatoxin contamination than Florunner in these tests. Significant positive correlations were observed between aflatoxin contamination and leaf temperature and between aflatoxin contamination and visual stress ratings. Leaf temperature and visual stress ratings are less variable and less expensive to measure than aflatoxin contamination. Leaf temperature and visual stress ratings maybe useful in indirectly selecting for reduced aflatoxin contamination in breeding populations.

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.

Apurinic/apyrimidinic (AP) endonuclease Ape1 is a key enzyme in the mammalian base excision repair pathway that corrects AP sites in the genome. Ape1 cleaves the phosphodiester bond immediately 5′ to AP sites through a hydrolytic reaction involving a divalent metal co-factor. Here, site-directed mutagenesis, chemical footprinting techniques, and molecular dynamics simulations were employed to gain insights into how Ape1 interacts with its metal cation and AP DNA. It was found that Ape1 binds predominantly to the minor groove of AP DNA, and that residues R156 and Y128 contribute to protein-DNA complex stability. Furthermore, the Ape1-AP DNA footprint does not change along its reaction pathway upon active-site coordination of Mg2+ or in the presence of DNA polymerase beta (polβ), an interactive protein partner in AP site repair. The DNA region immediately 5′ to the abasic residue was determined to be in close proximity to the Ape1 metal-binding site. Experimental evidence is provided that amino acid residues E96, D70, and D308 of Ape1 are involved in metal coordination. Molecular dynamics simulations, starting from the active site of the Ape1 crystal structure, suggest that D70 and E96 bind directly to the metal, while D308 coordinates the cation through the first hydration shell. These studies define the Ape1-AP DNA interface, determine the effect of polβ on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics.

The Vikings initially ventured into the Irish Sea as raiders and took, from the monasteries and other rich centres, wealth in the form of goods and slaves. In the course of the tenth century, however, they became permanently established and founded and developed the first towns in Ireland, often under sufferance from the local population. From these towns they controlled the trade routes along the western coasts of Europe through the Irish Sea — routes that brought luxury goods to both the North and the South. The increasing economic power of the Irish towns was one of the factors that led to the English conquest of Ireland in 1170.

There are two major apurinic/apyrimidinic (AP) endonuclease/3′-diesterase families designated after the Escherichia coli proteins exonuclease III (ExoIII) and endonuclease IV (EndoIV). These repair proteins function to excise mutagenic and cytotoxic AP sites or 3′-phosphate/phosphoglycolate groups from DNA. In mammals, the predominant repair endonuclease is Ape1, a homolog of ExoIII, whereas a mammalian homolog to EndoIV has not been identified to date. We have identified a human protein termed Ape2 that represents a subclass of the ExoIII family (exhibiting highest similarity to the Saccharomyces cerevisiae ETH1/APN2 gene product) and maintains many of the essential functional residues of the ExoIII-like proteins. The human protein is 518 amino acids with a predicted molecular mass of 57.3 kDa and a pI of 8.65. Unlike Ape1, this protein exhibited only weak ability to complement the repair defects of AP endonuclease/3′-repair-defective bacteria and yeast. Similarly, a weak, but specific, DNA-binding and incision activity for abasic site-containing substrates was observed with partially purified Ape2 protein. APE2 is located on the X chromosome at position p11.21 and consists of six exons. The transcript for APE2 is ubiquitously expressed, suggesting an important function for the encoded protein. An Ape2 green fluorescent fusion protein localized predominantly to the nucleus of HeLa cells, indicating a nuclear function; this localization was dependent on the C-terminal domain. We discuss our results in the context of the evolutionary conservation of the AP endonuclease families and their divergent activities and biological contributions. Environ. Mol. Mutagen. Environ. Mol. Mutagen. 36:312–324, 2000. Published 2000 Wiley-Liss, Inc.

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.

Immunostaining techniques are commonly employed to determine the temporal and spatial patterns of cellular components in in situ preparations of cells and tissues. Usually, cells are formalin-fixed, permeabilized with nonionic detergents, and probed with specific antibodies. The incorporation of a sodium dodecyl sulfate (SDS) treatment after chemical crosslinking has been shown to improve the immunodetection of some cytosolic and cell surface antigens. By incorporating an SDS treatment after crosslinking, we report a significant improvement in the detection of two nuclear antigens (i.e., the DNA binding proteins apurinic/apyrimidinic endonuclease and DNA polymerase-β) and bromodeoxyuridine-tagged DNA by indirect immunofluorescence of whole cells. In bromodeoxyuridine-tagged DNA, the improvement in detection after an SDS treatment was observed only after long incorporation protocols (>48 hr) and, interestingly, it was more pronounced in cultured human foreskin keratinocytes than in bovine aorta endothelial cells. In addition, the SDS treatment proved in these studies to be superior to the standard Triton X-100 permeabilization. SDS thus provides a potential means to visualize previously undetectable or poorly detectable nuclear antigens. (J Histochem Cytochem 47:1095–1100, 1999)

Stone composition, as reflected in radiographic appearance, is important to help choose between SWL and percutaneous/endoscopic procedures. Predicting a stone's composition accurately from a plain radiograph would be a useful tool in clinical decision-making. However, the ability of physicians to predict composition has not been adequately assessed. A prospective study was designed to quantify the accuracy of a panel of physicians who routinely deal with stones in classifying stone composition solely from radiographs.A panel of six members was created to review 100 plain-film radiographs from patients with renal stones of known composition. The panel consisted of two urologists, two radiologists, and two nephrologists, all of whom have expertise in stone disease. If the composition guessed was at least 40% of the total stone composition, the response was deemed correct.Overall, there was an average 39% correct response score among the six panelists. When the stones were divided by size, 35% were1 cm, and 65% were larger. The accuracy of chemical composition determination did not improve with greater stone size, nor was there a difference in accuracy for pure and mixed stones. The most frequently misclassified stone was calcium phosphate, with only 14% being correctly diagnosed.With a random sampling of plain radiographs, a panel of physicians specializing in stone disease correctly diagnosed the composition of renal calculi less than half of the time without being given clinical information.

DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.

Background Ifosfamide and cisplatin are active agents that are currently used in the treatment of osteosarcoma. Nephrotoxicity has been reported following their use in combination and alone. This study evaluates renal function in children and adolescents (median age 16 years) at least 3 months following completion of a chemotherapy regimen which included 54 g/m2 ifosfamide, 360 mg/m2 cisplatin, doxorubicin, and high-dose methotrexate. Procedure Mean glomerular filtration rate (GFR) was determined by inulin or iothalamate clearance; proximal tubular function was evaluated by measuring fractional excretion of glucose (FEglu), tubular maximum phosphate reabsorption per GFR (TMP/GFR), FE of urate, and 24-hour amino acid excretion. Distal tubular function was evaluated by 24-hour urinary calcium, FE of magnesium, and urinary osmolality after water deprivation. Twenty-four-hour urinary protein excretion was measured. Results The mean GFR was 97 ml/min/l.73 m2. Although 10 of 24 patients had GFRs lower than normal, the lowest value was only 22% below the lower limit of normal and would not account for any clinical compromise. Proximal tubular function evaluation revealed normal FEglu, normal mean TMP/GFR values, and high FE of urate (15.7%). Two of twenty-four patients were shown to have mild generalized aminoaciduria. Distal tubular function evaluation showed normal 24-hour urinary calcium levels (mean 3.4 mg/kg) and FE of magnesium as well as normal urinary osmolality. Twenty-four-hour urinary protein excretion was normal in all patients. Conclusions The lack of clinically significant renal abnormalities observed in patients who received combination chemotherapy with ifosfamide and cisplatin for osteosarcoma is encouraging for future osteosarcoma protocol development. Med. Pediatr. Oncol. 32:93–96, 1999. © 1999 Wiley-Liss, Inc.

In vitro biochemical studies indicate that Ape1 is the major mammalian enzyme responsible for repairing abasic lesions in DNA and a significant factor in the processing of specific 3′-replication-blocking termini. Toward addressing the role of Ape1 in cellular resistance to specific DNA-damaging and anticancer agents, we constructed a chinese hamster ovary (CHO) cell line, AA8-Ape1, that exhibits a 7-fold higher Ape1-dependent nuclease activity; this overexpression is abolished upon exposure to tetracycline (Tc). In comparison to the AA8 parental control, our data indicates that Ape1 activity is not rate-limiting for the repair of cytotoxic damages induced by the alkylating agent methyl methanesulfonate (MMS), the oxidizing agent hydrogen peroxide (H2O2), or ionizing radiation (IR). AA8-Ape1 cells did exhibit increased resistance to bleomycin following a chronic 3-day exposure, but not to more acute challenges of 1 h. Most notably, the AA8-Ape1 line displayed ∼1.7-fold elevated resistance to the replication-blocking nucleoside analog dioxolane cytidine (L-OddC); this improved resistance was abrogated by the addition of Tc to the medium. These studies demonstrate that Ape1 is not rate-limiting in the repair of MMS- or H2O2-induced DNA damage, that Ape1 may dictate the sensitivity of bleomycin, depending on dosing scheme, and for the first time, that Ape1 can influence cellular resistance to the anticancer/antiviral antimetabolite L-OddC.

Maize samples were collected from nine Grain Marketing Board (G.M.B) centers in Zimbabwe during the 1991 harvest season. A further 47 samples collected directly from farmers and from the G.M.B., centers in Chinhoyi and Kwekwe during the 1992 harvest season. These samples were analyzed mycologically and the predominant flora was Fusarium although Penicillium, Nigrospora, Aspergillus and Chaetomium could be isolated from some samples. From the first nine samples studied, F. verticillioides and F. subglutinans were isolated in almost equal proportions on samples from the central and the south of the country whereas only F. verticillioides was isolated on the samples from the north. The subsequent study demonstrated that there was a greater fungal diversity in samples from North (Mashonaland West) than samples from the South (Midlands area) with species of Nigrospora, Chaetomium, Acremonium and Diplodia occurring in significant numbers. From a total of 2821 fungal isolates obtained from all the maize samples analyzed, 1485 (53%) were found to belong to the liseola section of Fusarium. The ability of these isolates to produce the mycotoxins zearalenone, moniliformin and fumonisin B1 was tested using a simplified TLC Agar plate method. Out of the 886 isolates tested, only one produced all the three mycotoxins simultaneously whilst most produced fumonisin B1 and/or moniliformin. Only nine isolates produced zearalenone.

The effects of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide HCl (SR 141716A), a specific cannabinoid receptor antagonist, were assessed in the dog static ataxia test after either acute treatment with two cannabinoid receptor agonists, delta9-tetrahydrocannabinol and arachidonylethanolamide (anandamide), or chronic treatment with delta9-tetrahydrocannabinol. As previously reported, acute intravenous (i.v.) injected delta9-tetrahydrocannabinol produced dose-dependent cannabinoid effects, including marked static ataxia, prancing, loss of muscle tone, and incoordination. The behavioral profile of anandamide was distinctly different in that it produced a loss of muscle tone and considerable sedation with little static ataxia, prancing, or incoordination. Despite these qualitative differences between the two agonists, SR 141716A blocked the acute behavioral effects of both drugs indicating a cannabinoid receptor mechanism of action. Interestingly, SR 141716A was able to precipitate a withdrawal syndrome in delta9-tetrahydrocannabinol-tolerant dogs, but failed to produce any observable effects in dogs receiving chronic vehicle injections. Acute toxicity caused by anandamide, which was not blocked by SR 141716A, precluded conducting dependence studies with this drug. The delta9-tetrahydrocannabinol precipitated withdrawal syndrome included diarrhea, vomiting, excessive salivation, decreases in social behavior, and increases in restless behavior and trembling. This is the first demonstration of a precipitated withdrawal syndrome in a non-rodent species.

Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication. We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product. Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair. Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity. Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development. HEX1 gene equivalents were found in all vertebrates examined. The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors.

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a

The administration of ammonium chloride or of sodium or potassium bicarbonate has marked effects on the development of polycystic kidney disease (PKD) in Han:SPRD rats. Because of the possibility that these effects are mediated by changes in redox metabolism, the aim of this study was to determine whether depletion of glutathione, the most abundant and important cellular thiol and scavenger of reactive oxygen species, would affect the development of PKD in this animal model. +/+ and cy/+ Han:SPRD rats were treated with: (1) L-buthionine(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the synthesis of glutathione; (2) glutathione monoethyl ester (GME), a compound that is known to increase the intracellular levels of glutathione; or (3) BSO and GME. Treatment with these drugs was started at 3 wk of age, and the animals were killed at 6 or 8 wk of age. Renal levels of oxidized glutathione were significantly higher in cy/+ than in +/+ rats, whereas no significant differences in reduced glutathione were detected. The administration of BSO caused a marked reduction in the levels of glutathione. The administration of GME caused a significant increase in the levels of glutathione at 2 h, but not at 12 h, after the administration. The increase in the renal levels of glutathione 2 h after the administration of GME was less in the rats treated with BSO than in the rats not receiving this drug, indicating that in part the increase in glutathione level was due to de novo synthesis. BSO-induced glutathione depletion was accompanied by a marked aggravation of the renal cystic disease, as reflected by kidney weights, histological scores, and plasma urea concentrations. However, the administration of GME did not lessen the cystic disease and did not reverse the effects of BSO. The transient effect of GME administration and the simultaneous increases in the levels of cysteine and oxidized glutathione, in addition to reduced glutathione, may explain the lack of protection by GME. These data support the notion that changes in redox metabolism may affect the development of PKD.

The interactions between dopamine, cocaine, cocaethylene, and ethanol were studied in Swiss–Webster mice. The loss of the righting reflex (LORR) was used as a measure of CNS depression. Animals were injected intraperitoneally (IP) with ethanol (4.0 g/kg), which caused a LORR. Immediately upon regaining of the righting reflex, mice were injected intracerebroventricularly (ICV) with saline, dopamine (0.1, 0.5, or 1.0 μmol/kg), cocaine (1, 15, or 25 μmol/kg), or cocaethylene (1, 15, or 25 μmol/kg). In the presence of systemic ethanol, all three compounds produced CNS depression in a dose-dependent manner. The dopamine D 2 -receptor antagonist sulpiride and the D 1 -receptor antagonist fluphenazine were given acutely ICV with dopamine in the presence of systemic ethanol to examine whether these antagonists could block the return to the LORR produced by dopamine. Sulpiride, however, actually enhanced the interaction between ethanol and dopamine in a dose-dependent manner as measured by the LORR; fluphenazine neither blocked nor enhanced the effect of dopamine in the presence of systemic ethanol. In addition, these antagonists had no effect on cocaine- and cocaethylene-induced CNS depression in the presence of systemic ethanol. The results of this study showed that the neurotransmitter dopamine and both cocaine and cocaethylene can promote further CNS depression in the presence of systemic ethanol, and that dopamine was significantly more potent than cocaine and cocaethylene as measured by the return to the LORR.

Changes in oral temperature can influence taste perception, indicating overlap among mechanisms for taste and oral somesthesis. Medullary gustatory neurons can show cosensitivity to temperature, albeit how these cells process combined taste and thermal input is poorly understood. Here, we electrophysiologically recorded orosensory responses (spikes) from 39 taste-sensitive neurons in the nucleus tractus solitarii of anesthetized mice during oral delivery of tastants adjusted to innocuous cool (16 and 18°C), room (22°C, baseline), and warm (30 and 37°C) oral temperatures. Stimuli included (in mM) 100 sucrose, 30 NaCl, 3 HCl, 3 quinine, an umami mixture, and water. Although cooled water excited few cells, water warmed to 30 and 37°C significantly excited 33% and 64% of neurons, respectively. Warmth induced responses of comparable magnitude to room temperature tastants. Furthermore, warming taste solutions influenced the distribution of gustatory responses among neurons and increased ( P < 0.05) neuronal breadth of tuning across taste qualities. The influence of warmth on response magnitude was stimulus specific. Across neurons, warming facilitated responses to sucrose and umami in a superadditive manner, as these responses exceeded ( P < 0.05) the arithmetic sum of activity to warming alone and the taste stimulus tested at room temperature. Superadditive increases ( P < 0.05) in responding were also noted in some cells for warmed HCl. Yet warming induced only simple additive or subtractive effects on responses to quinine and NaCl. Data show temperature is a parameter of gustatory processing, like taste quality and concentration, in medullary circuits for taste.

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.

UnlabelledReduction and oxidation (redox) chemistry is increasingly implicated in cancer pathogenesis. To interrogate the redox status of prostate tumors noninvasively, we developed hyperpolarized [1-(13)C]dehydroascorbate ((13)C-DHA), the oxidized form of vitamin C, as an MR probe. In a model of transgenic adenocarcinoma of the mouse prostate (TRAMP), increased reduction of hyperpolarized (13)C-DHA to vitamin C was observed in tumor, as compared with normal prostate and surrounding benign tissue. We hypothesized that this difference was due to higher concentrations of glutathione and increased transport of hyperpolarized (13)C-DHA via the glucose transporters (GLUT1, GLUT3, and GLUT4) in TRAMP tumor. To test these hypotheses, hyperpolarized (13)C-DHA MR spectroscopy (MRS) and (18)F-FDG PET were applied as complementary technologies in the TRAMP model.MethodsLate-stage TRAMP tumors (>4 cm(3)) were studied at similar time points (MR studies conducted < 24 h after PET) in fasting mice by (18)F-FDG PET and hyperpolarized (13)C-DHA MR imaging on a small-animal PET/CT scanner and a (1)H/(3)C 3-T MR scanner. PET data were processed using open-source AMIDE software to compare the standardized uptake values of tumor with those of surrounding muscle, and (13)C-DHA MRS data were processed using custom software to compare the metabolite ratios (vitamin C/[vitamin C + (13)C-DHA]). After in vivo studies, the tumor glutathione concentrations were determined using a spectrophotometric assay, and thiol staining was performed using mercury orange. Real-time polymerase chain reaction was used to evaluate the relevant transporters GLUT1, GLUT3, and GLUT4 and vitamin C transporters SVCT1 and SVCT2. GLUT1 was also evaluated by immunohistochemistry.ResultsThe average metabolite ratio was 0.28 ± 0.02 in TRAMP tumor, versus 0.11 ± 0.02 in surrounding benign tissue (n = 4), representing a 2.5-fold difference. The corresponding tumor-to-nontumor (18)F-FDG uptake ratio was 3.0. The total glutathione was 5.1 ± 0.4 mM in tumor and 1.0 ± 0.2 mM in normal prostate, whereas reduced glutathione was 2.0 ± 0.3 mM and 0.8 ± 0.3 mM, respectively, corresponding to a 2.5-fold difference. In TRAMP tumor, mercury orange staining demonstrated increased thiols. Real-time polymerase chain reaction showed no significant difference in GLUT1 messenger RNA between TRAMP tumor and normal prostate, with immunohistochemistry (anti-GLUT1) also showing comparable staining.ConclusionBoth hyperpolarized (13)C-DHA and (18)F-FDG provide similar tumor contrast in the TRAMP model. Our findings suggest that the mechanism of in vivo hyperpolarized (13)C-DHA reduction and the resulting tumor contrast correlates most strongly with glutathione concentration. In the TRAMP model, GLUT1 is not significantly upregulated and is unlikely to account for the contrast obtained using hyperpolarized (13)C-DHA or (18)F-FDG.