Strong genetic influences on measures of behavioral-regulation among inbred rat strains.

A fundamental challenge for any complex nervous system is to regulate behavior in response to environmental challenges. Three measures of behavioral-regulation were tested in a panel of eight inbred rat strains. These measures were: (1) sensation seeking as assessed by locomotor response to novelty and the sensory reinforcing effects of light onset, (2) attention and impulsivity, as measured by a choice reaction time task and (3) impulsivity as measured by a delay discounting task. Deficient behavioral-regulation has been linked to a number of psychopathologies, including ADHD, Schizophrenia, Autism, drug abuse and eating disorders. Eight inbred rat strains (August Copenhagen Irish, Brown Norway, Buffalo, Fischer 344, Wistar Kyoto, Spontaneous Hypertensive Rat, Lewis, Dahl Salt Sensitive) were tested. With n = 9 for each strain, we observed robust strain differences for all tasks; heritability was estimated between 0.43 and 0.66. Performance of the eight inbred rat strains on the choice reaction time task was compared to the performance of outbred Sprague Dawley (n = 28) and Heterogeneous strain rats (n = 48). The results indicate a strong genetic influence on complex tasks related to behavioral-regulation and indicate that some of the measures tap common genetically driven processes. Furthermore, our results establish the potential for future studies aimed at identifying specific alleles that influence variability for these traits. Identification of such alleles could contribute to our understanding of the molecular genetic basis of behavioral-regulation, which is of fundamental importance and likely contributes to multiple psychiatric disorders.

Effect of thymine glycol on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II.

Thymine glycols are formed in DNA by exposure to ionizing radiation or oxidative stress. Although these lesions are repaired by the base excision repair pathway, they have been shown also to be subject to transcription-coupled repair. A current model for transcription-coupled repair proposes that RNA polymerase II arrested at a DNA lesion provides a signal for recruitment of the repair enzymes to the lesion site. Here we report the effect of thymine glycol on transcription elongation by T7 RNA polymerase and RNA polymerase II from rat liver. DNA substrates containing a single thymine glycol located either in the transcribed or nontranscribed strand were used to carry out in vitro transcription. We found that thymine glycol in the transcribed strand blocked transcription elongation by T7 RNA polymerase approximately 50% of the time but did not block RNA polymerase II. Thymine glycol in the nontranscribed strand did not affect transcription by either polymerase. These results suggest that arrest of RNA polymerase elongation by thymine glycol is not necessary for transcription-coupled repair of this lesion. Additional factors that recognize and bind thymine glycol in DNA may be required to ensure RNA polymerase arrest and the initiation of transcription-coupled repair in vivo.

Using the Microcyte flow cytometer to monitor cell number, viability, and apoptosis in mammalian cell culture.

The Microcyte is a novel, portable flow cytometer based on diode laser technology whose use has been established for yeast and bacterial analysis. We present data that demonstrate its suitability for routine mammalian cell counting and viability determination. To extend its range of applications in the field of animal cell culture biotechnology, a test to determine the number of apoptotic cells present has been developed for use with the instrument. Apoptosis was induced in hybridoma cell cultures by treatment with camptothecin. Apoptotic cells were labeled with biotinylated Annexin V and then visualized using a streptavidin-allophycocyanin conjugate. Their numbers were counted, and the cell size of the apoptotic cell population was determined using the Microcyte.

p53-dependent global genomic repair of benzo[a]pyrene-7,8-diol-9,10-epoxide adducts in human cells.

The global genomic repair of DNA adducts formed by the human carcinogen (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) has been studied by 32P-postlabeling in human fibroblasts in which p53 expression can be regulated. At low BPDE adduct levels (10-50 adducts/10(8) nucleotides), repair was rapid and essentially complete within 24 h in p53+ cells, whereas no repair was detected within 72 h in similarly treated p53- cells. At 10-fold higher BPDE adduct levels, repair under both conditions was rapid up to 8 h, after which a low level of adducts persisted only in p53- cells. These results demonstrate a dependence on p53 for the efficient repair of BPDE adducts at levels that are relevant to human environmental exposure and, thus, have significant implications for human carcinogenesis.

Relationship between cell size, cell cycle and specific recombinant protein productivity.

Centrifugal elutriation was used to produce cell cycle enrichedfractions of four commercially relevant recombinant cell lines,chosen to allow for variation in properties due to construct,expression system and parent cell type, from normally growingheterogeneous batch cultures. As these fractions had identicalculture histories and had not been subjected to any insult orstress which was likely to have adversely affected cellularmetabolism, they were ideal for further study of cellularproperties. Specific productivity, cell size and cell cyclestate of replicate elutriated fractions were measured for eachcell line. Results showed that cell size was the major cellulardeterminant of productivity for all cell lines examined. Productformation was not restricted to any particular cell cycle phaseand in all cases, production occurred irrespective of cell cyclephase. Specific productivity was lowest when the majority ofcells in the fraction were G(1), intermediate when themajority of cells in the fraction were S phase and greater whenthe majority of cells in the fraction were in G(2)/M. However, the evidence suggests that size is the major cellulardeterminant of productivity; the apparent relationship betweencell cycle and productivity is secondary and can simply beascribed to the increasing size of cells as they progress thoughthe cell cycle. Thus, in addition to cell density and viabilitycell size is the cellular parameter which should be incorporatednot only into mathematical models of recombinant mammalian cellproduction processes but also into process monitoring andcontrol strategies.

Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay.

Apoptosis is a form of cell death in which the dying cell plays an active part in its demise. At the morphological level, it is characterised by cell shrinkage rather than the swelling seen in necrotic cell death. In cell culture, apoptosis limits the yield of economically and medically important products, and can result in synthesis of imperfect molecules. Therefore, this process must be identified, monitored and fully understood, so that a means to regulate it can be developed. We have developed a new automatic image analysis assay for detecting apoptosis in animal cell culture on the basis of the annexin-V affinity assay. The results of this assay were compared with data generated by flow cytometry and manual scoring. All three methods were found to correspond well but image analysis like flow cytometry offers operator-independent results, and can be used as a tool for rapid monitoring of viable cell number, apoptosis and necrosis in animal cell culture. Furthermore, reduction in cell size was measured and was found to precede the appearance of phosphatidylserine on the cell surface.

Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-hydroxydeoxyguanosine and putative intrastrand cross-links.

The role of metal ion-DNA interactions in the Fenton reaction-mediated formation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA and pBluescript K+ plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity for DNA and in the spectrum of oxidative DNA damage they induce in Fenton reactions. EDTA was included in these incubations according to two different strategies; the first (strategy 1) in which DNA and metal ions were mixed prior to the addition of EDTA, the second (strategy 2) in which EDTA and metal ions were mixed prior to the addition of DNA. The formation of the putative intrastrand cross-links, monitored by 32P-postlabelling, was not affected by the addition of between 10 microM and 5 mM EDTA to the copper(II) Fenton reaction according to strategy 1. In contrast, the level of cross-links declined significantly upon inclusion of 20 microM EDTA and above when added according to strategy 2. Similarly, formation of these lesions declined in the iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA when added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(II) Fenton reaction declined equally with both strategies with up to 25 mM EDTA. The formation of single- and double-strand breaks was investigated in plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of linear DNA in the iron(II) Fenton reaction decreased dramatically upon inclusion of EDTA according to strategy 2, while no such decline was observed using strategy 1. In contrast, the formation of linear DNA in the copper(II) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the formation of open-circular DNA was also observed upon inclusion of EDTA according to both strategies; however this decrease occurred at a lower EDTA concentration in strategy 2 (100 microM) compared to strategy 1 (200 microM), and the level of open-circular DNA reached a lower level (8. 5% compared to 24.2%). The nickel(II) Fenton reaction generated only open-circular DNA, and this was completely inhibited upon addition of 25 microM EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(II) Fenton reactions when EDTA was added according to strategy 2 than according to strategy 1. These results suggest that a site-specific mechanism is involved in the formation of double-strand breaks and, to a lesser extent, 8-OHdG and the putative intrastrand cross-links, while the formation of single-strand breaks is more likely to involve generation of hydroxyl radicals in solution.

The role of the cell cycle in determining gene expression and productivity in CHO cells.

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.

Comparison of the formation of 8-hydroxy-2'-deoxyguanosine and single- and double-strand breaks in DNA mediated by fenton reactions.

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone. Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions. Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

Generation of putative intrastrand cross-links and strand breaks in DNA by transition metal ion-mediated oxygen radical attack.

Generation of putative intrastrand cross-links and strand breaks was investigated in salmon sperm DNA exposed to Fenton-type oxygen radical-generating systems. 32P-Postlabeling analysis of DNA treated with hydrogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(II), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radical-mediated oxidative DNA lesions. The copper Fenton system generated the highest total yield of these DNA lesions (75.6 per 10(8) nucleotides), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (21.7), and vanadium (17.1). Two spots, common to all these Fenton systems, were the major oxidation products in each case. Similar Fenton-type treatment of the purine dinucleotides dApdG and dApdA resulted in products that were chromatographically identical on anion-exchange TLC and on reverse-phase HPLC to the two major products generated in DNA. These results extend our earlier studies suggesting that these products were the result of a free radical-mediated intrastrand cross-linking reaction. Incubations involving cadmium(II), chromium(III), or zinc(II) ions with hydrogen peroxide did not generate DNA oxidation products at levels greater than in incubations with hydrogen peroxide alone. Generation of the putative intrastrand cross-links increased in a concentration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) ions. However, in experiments with copper, iron, or vanadium ions, maximum levels were obtained at 250, 150, and 150 microM, respectively, and the yield declined with higher concentrations of these three metal ions. Agarose gel electrophoresis demonstrated extensive DNA strand breakage with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results demonstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independent mechanisms.

A rapid method for evaluation of cell number and viability by flow cytometry.

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light.

Gastrointestinal and nutritional sequelae of bone marrow transplantation.

The nature of the gastrointestinal injury following bone marrow transplantation and its clinical and nutritional sequelae are poorly defined. Prospective assessments of gastrointestinal function, nutritional status, and wellbeing were therefore carried out in 47 consecutive patients (28 males, 19 females; mean age 8.4 years) undergoing bone marrow transplant. 31 diarrhoeal episodes (median duration 9.5 days) occurred in 27 patients at a median of 10 days after transplantation. Ninety one per cent of episodes were associated with protein losing enteropathy. Protein losing enteropathy was more severe in graft-versus-host disease (GVHD) comparing with other causes. It led to a substantial fall in serum albumin and there was a negative correlation between faecal alpha 1-antitrypsin concentrations and serum albumin. Transient pancreatic insufficiency developed in 18 patients, and pancreatitis in one. Intestinal permeability was normal in 12 patients who had no diarrhoea during the conditioning treatments. Diarrhoeal patients had a significantly greater decrease in nutritional status and wellbeing than patients without diarrhoea. Gastrointestinal injury following bone marrow transplantation is thus complex. Severe protein losing enteropathy in this context suggests the presence of GVHD.

Effects of sodium bicarbonate and sodium chloride on the elimination of etorphine in equine urine.

The combination of large doses of sodium bicarbonate and the potent narcotic, etorphine, has reportedly been given to racehorses in attempts to improve their performance and also to "mask" the presence of etorphine in urine samples. The increased urinary output and pH associated with sodium bicarbonate (approximately 500 g) administration may reduce the urinary concentration of etorphine, making it more difficult to detect. Our experiment was designed to examine the effects of this combination. Six Thoroughbred horses were used in a latin-square design with three horse pairs and three treatments consisting of the following: etorphine (20 micrograms), etorphine (20 micrograms) plus sodium bicarbonate (1.0 g/kg), and etorphine (20 micrograms) plus sodium chloride (0.7 g/kg). Sodium chloride was used to distinguish between the urinary alkalinizing effects of sodium bicarbonate and the diuretic effects associated with the large electrolyte load. Venous blood and urine samples were collected prior to and for 24 h post-treatment. Sodium bicarbonate produced a significant metabolic alkalosis and an increase in urine pH. Both sodium bicarbonate and sodium chloride produced a profound diuresis. After sodium bicarbonate and sodium chloride treatments, the urinary concentration of etorphine, measured by radioimmunoassay (RIA), was reduced and in some cases could not be detected. Extraction of the urine samples, prior to RIA analysis, increased the sensitivity of the assay and in most cases gave a positive result. We conclude that the coadministration of etorphine and sodium bicarbonate or sodium chloride can make the detection of etorphine more difficult because of the dilutional effects associated with the administration of a large electrolyte load.

Effects of sodium bicarbonate on fluid, electrolyte and acid-base balance in racehorses.

Sodium bicarbonate given by nasogastric tube has been used by some trainers as the key ingredient in a 'milkshake'. It has been suggested that such treatment given 3-5 h prior to racing may enhance a horse's racing performance by increasing the blood buffering capacity and enhancing lactate clearance from skeletal muscle, thereby delaying the onset of fatigue. Several experiments were conducted to examine the effects on fluid, electrolyte and acid-base values of 0.5 g kg-1 dose of sodium bicarbonate, were examined. The effects of fasting, the simultaneous administration of glucose (0.5 g kg-1) or the withholding of water were also examined to determine whether they influenced the uptake and elimination of sodium bicarbonate. Six Thoroughbred horses were used, each wearing a urine and faecal collection harness. Prior to sodium bicarbonate administration, venous blood, urine and faecal samples were collected for 24 h to establish control values. After administration of sodium bicarbonate (0.5 g kg-1) in 2 l of water, samples were collected at various times for up to 46 h. There were significant increases in water consumption, from 0.5-2.3 l h-1 at 2 h post-administration. Urine output increased by approximately three fold and did not return to control levels until 18 h post-administration. Urinary sodium concentration increased from 95 +/- 16 mmol l-1 (mean +/- SEM) to peak values of 349 +/- 12 mmol l-1 at 12 h. In the 24 h after sodium bicarbonate administration, approximately 80% of the sodium intake (NaHCO3+feed) was excreted in the urine. There was no significant change in the total urinary potassium and chloride excretion. Faecal water content did not change following sodium bicarbonate administration, but there was an increase in faecal sodium content. The mean increase in venous blood bicarbonate concentration was 7.6 +/- 0.4 mmol l-1 after the 0.5 kg-1 dose. Water deprivation for 6 h after sodium bicarbonate administration, fasting or the co-administration of glucose did not affect the peak blood bicarbonate concentration or the time to peak concentration. However, the withholding of water did result in a faster rate of decrease in blood bicarbonate concentration when water was resupplied.

Impaired bactericidal activity of PMN from two brothers with necrotizing ulcerative gingivo-periodontitis.

The purpose of this study was to investigate the pathogenesis of necrotizing ulcerative gingivo-periodontitis (ANUP) diagnosed in two brothers, age 9 (ANUP1) and 14 (ANUP2) from rural Egypt. Complete blood count, differential and blood chemistry were within normal limits for both brothers and they were not malnourished. The phagocytosis and killing function of their polymorphonuclear leukocytes (PMN) towards four bacterial species were assessed using a fluorochrome microassay. The selection of bacterial species was based on preliminary microbiological results in early onset periodontitis in Egypt. Fluorochrome-labeled Prevotella intermedia, Peptostreptococcus micros, Campylobacter rectus, and Porphyromonas gingivalis were pre-opsonized with ANUP serum and added to PMN from both ANUP patients, as well as PMN from three sex-matched and two sex- and age-matched healthy Egyptian control (CTL) subjects. We found significant depressions (P < 0.05) in PMN phagocytosis and killing of C. rectus and P. intermedia by ANUP1 and ANUP2, when compared to all CTL PMN. An assessment of the Gram-negative subgingival microflora present in both ANUP patients (in colony forming unit percent of total CFU recovered) (CFU %) revealed the presence of P. intermedia (ANUP1, 41.7 CFU %; ANUP2, 14.8 CFU %), Fusobacterium nucleatum (ANUP1, 3.6 CFU %; ANUP2, 48.1 CFU %), and Veillonella spp. (ANUP1, 18.2 CFU %; ANUP2, 18.5 CFU %). Spirochetes were also observed in cytocentrifuged, Gram-stained plaque from both ANUP patients. The predominant Gram-positive bacterial species recovered from both NUG1 and NUG2 was Streptococcus morbillorum.

Use of the Syrian golden hamster for the induction of intraoral abscesses by sutures contaminated with human subgingival plaque.

Analysis of normal oral flora in 150 cheek pouches of hamsters (Mesocricetus auratus) defined the microbial working environment and demonstrated the absence of human oral black-pigmented bacteria. Silk sutures saturated with Porphyromonas gingivalis, Prevotella intermedia or subgingival plaque were used to close wounds made in hamster's cheek pouches. Abscesses were formed when sutures had solitary P. gingivalis or other bacteria mixed with P. gingivalis or when P. intermedia was mixed with other bacteria besides P. gingivalis. A concentration of black-pigmented bacteria emanating from 3 x 10(5) colony-forming units/inoculum was required for abscess formation. Six abscesses (14.3%) were developed in association with the presence of other odontopathic bacteria, primarily Fusobacterium nucleatum and Actinomyces viscosus. The hamster cheek pouch with iatrogenic wounds closed with plaque-impregnated sutures is a novel and effective model to study the pathology of wound infections and virulence of human subgingival organisms.

Pathological changes in the rabbit ileal loop model caused by Campylobacter jejuni from human colitis.

Four strains of Campylobacter jejuni isolated from children with inflammatory diarrhoea were assayed in the rabbit ileal loop model of infectious diarrhoea. All caused inflammatory reactions with severe macroscopic and microscopic damage in infected rabbit ileal tissue similar to that observed in the patients by endoscopy and histological analysis of colonic biopsies. Haemoglobin and other proteins were observed in loop fluids, consistent with leakage of serum from damaged mucosa. Loop fluids also contained significant bicarbonate concentrations, indicative of an active secretory component similar to that in control loops inoculated with cholera toxin. However, although three of the four clinical strains produced small amounts of a protein immunologically related to cholera toxin in vitro, none such was detected in either tissues or fluids of infected ileal loops. We propose instead that host-derived mediators of secretion may be important in pathogenesis. A mutant strain of C. jejuni with impaired motility, obtained from the National Collection of Type Cultures, did not induce tissue damage or fluid secretion in rabbit ileal loops.

Effects of sodium bicarbonate on cardiorespiratory measurements and exercise capacity in thoroughbred horses.

We examined the effects of sodium bicarbonate in 6 Thoroughbred horses during submaximal and maximal treadmill exercise. Cardiorespiratory function was assessed together with the effect on exercise capacity by determining the run time to fatigue at maximal intensities. To discriminate between sodium bicarbonate's alkalinising effects and the fluid shifts that could result from the high osmotic load, we administered an equimolar solution of sodium chloride as a control. The horses were given sodium bicarbonate (1 g/kg bwt) or an equivalent number of moles of sodium chloride by nasogastric tube. Arterial blood samples were collected before exercise and 5 h after treatment, resulting in mean standard bicarbonate values of 39.6 mmol/l in horses treated with sodium bicarbonate compared with 24.2 mmol/l in horses that received saline. The horses were exercised on a treadmill at 40, 60 and 80% of their VO2max for 4, 2 and 2 mins respectively. The horses were walked for 3 mins and accelerated rapidly to a speed approximately equivalent to 110% VO2max and run until fatigued. The horses ran for 170 +/- 20 secs (mean +/- sem) after administration of sodium bicarbonate compared with 128 +/- 13 secs after receiving sodium chloride (P < 0.02). At rest and throughout submaximal and maximal exercise, the bicarbonate-treated horses had significantly lower arterial oxygen tensions and higher arterial carbon dioxide tensions. There were no differences in cardiac output, heart rate, oxygen uptake or carbon dioxide production between the saline and bicarbonate treatments.(ABSTRACT TRUNCATED AT 250 WORDS)