Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes.

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Melody recognition using three types of dichotic-pitch stimulus.

The recognition of 10 different 16-note melodies, constructed using either dichotic-pitch stimuli or diotic pure-tone stimuli, was measured. The dichotic pitches were created by placing a frequency-dependent transition in the interaural phase of a noise burst. Three different configurations for the transition were used in order to give Huggins pitch, binaural-edge pitch, and binaural-coherence-edge pitch. Forty-nine inexperienced listeners participated. The melodies evoked by the dichotic stimuli were consistently identified well in the first block of trials, indicating that the sensation of dichotic pitch was relatively immediate and did not require prolonged listening experience. There were only small improvements across blocks of trials. The mean scores were 97% (pure tones), 93% (Huggins pitch), 89% (binaural-edge pitch), and 77% (binaural-coherence-edge pitch). All pairwise differences were statistically significant, indicating that Huggins pitch was the most salient of the dichotic pitches and binaural-coherence-edge pitch was weakest. To account for these differences in salience, a simulation of lateral inhibition was applied to the recovered spectrum generated by the modified equalization cancellation model [J. F. Culling, A. Q. Summerfield, and D. H. Marshall, J. Acoust. Soc. Am. 103, 3509-3526 (1998)]. The height of the peak in the resulting "edge-enhanced" recovered spectrum reflected the relative strength of the different dichotic pitches.

Infant affective responses to mother's still face at 6 months differentially predict externalizing and internalizing behaviors at 18 months.

This study investigated (a) stability and change in infant affective responses to the still-face interaction, (b) whether maternal depression affected infant responses, and (c) whether responses to the still-face interaction predicted toddler problem behaviors. Infants (63 girls and 66 boys) of European American mothers (67 depressed and 62 nondepressed) were observed in the still-face interaction at 2, 4, and 6 months. Affect and gaze were coded on a 1-s time base. There were stable individual differences in gazing away and in rates of negative affect. Developmental change occurred only for gazing away, which increased. At 18 months, infants who failed to smile at 6 months in the still-face interaction showed more externalizing-type behaviors than did other toddlers. Infants who failed to cry at 6 months showed fewer internalizing-type behaviors. Mothers' current depressive symptoms and infants' earlier responses to the still-face interaction made independent, comparable contributions to problem behaviors at 18 months.

Oranges and lemons: clues to the taxonomy of Citrus from molecular markers.

Go into any grocery store and one is confronted with an array of Citrus fruit: oranges, grapefruit, mandarins (tangerines), lemons and limes. This is rich bounty for the shopper, but taxonomists are perplexed as to how to classify the various kinds of Citrus that have existed since antiquity. Now, thanks to new genetic and molecular biological techniques, the relationships between these fruit are being unraveled and show that there are probably only three true species.

Mothers' affective behavior with infant siblings: stability and change.

To evaluate within-family differences in maternal affective behavior toward siblings, face-to-face interactions were observed between 39 mothers and their firstborn and second-born infants at 2 months of age. Mother and infant affect was coded on a 1-s time base with behavioral descriptors. Mothers were more positive with second-born infants, and second-born infants were more positive than were firstborns. The siblings' affective behaviors were unrelated, but maternal positive affect was both moderately stable between siblings and correlated with each infant's affect. Thus, in the context of stable individual differences in maternal positive affect, siblings experienced unique affective interactions with their mothers as early as 2 months.

Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein.

During tomato fruit ripening, photosynthetically competent thylakoid membranes are broken down and replaced by membranous deposits of carotenoids. Few of the proteins involved in this transition have been identified. We have used chloroplast protein import assays as a means to identify two cDNAs that encode proteins destined for the developing chromoplast. One of the cDNAs had unexpected properties and its biological function has not been determined. However, the other cDNA encodes a plastid-localized low-MW heat shock protein (hsp). The steady-state level of RNA corresponding to this cDNA increased several-fold during tomato ripening, and the amount of RNA induced by heat stress increased dramatically during this process. These observations suggest a new role for this stress protein in protecting the plastid during the dismantling of the thylakoid membranes or during the buildup of carotenoids.

Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus.

Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of ``escape'' shoots produced. Although the use of a wild-type ``shooty'' Agrobacterium strain allowed relatively high frequencies of ß-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root. Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus.

Relationship between mitochondrial dysfunction and toxicity of propyl gallate in isolated rat hepatocytes.

The relationship between cytotoxicity and mitochondrial dysfunction caused by propyl gallate (PG) has been studied in hepatocytes freshly prepared from fasted rats. Hepatocytes isolated from fasted (18 h) rats were significantly more susceptible to the toxicity of PG than hepatocytes from fed rats. The addition of fructose (15 mM), an alternative carbohydrate source, to hepatocyte suspensions resulted in the prevention of PG (1 mM)-induced cell killing accompanied by decrease in intracellular ATP loss during a 3 h-incubation period. Despite this, fructose did not completely prevent an abrupt loss of intracellular glutathione caused by PG, but effectively inhibited the loss of protein thiol levels. Fructose elicited a concentration (0.5-20mM)-dependent protection against the cytotoxicity of 1.5 mM PG. The incubation of hepatocytes with sodium azide (4 mM), an inhibitor of oxidative phosphorylation, enhanced the toxicity induced by PG (1 mM), but coincubation with fructose delayed the onset of toxicity. Neither azide alone nor fructose plus azide did affect the cell viability during the incubation period. Furthermore, the addition of 2 mM salicylamide, nontoxic to hepatocytes during the incubation period, enhanced PG (1 mM)-induced cytotoxicity and decreased the loss of free PG. These results indicate that the onset of cytotoxicity caused by PG may depend on the intracellular energy status and that mitochondria are critical target for the compound. In addition, the toxicity caused by the inhibition of mitochondrial ATP synthesis is related to the concentration of PG remaining in cell suspensions.

Detection of cytosine methylation and mapping of a gene influencing cytosine methylation in the genome of Citrus.

A new method was developed to detect DNA methylation in the Citrus genome using random amplification coupled with restriction enzyme digestion. Genomic DNA from Citrus grandis (L.) Osb., Poncirus trifoliata (L.) Raf., and their F1 hybrid was amplified using 7 individual 10-mer random primers. Prior to amplification the DNA templates were digested with 2 pairs of restriction endonucleases (HpaII-MspI and (or) Sau3AI-NdeII) with different sensitivities to cytosine methylation and after PCR amplification their amplified products were further digested with the same enzymes. Using this method, it was possible to detect 28 methylation events involving 23 amplified bands with the 7 random primers and 2 pairs of enzymes. A methylation polymorphism was found at a Sau3AI site in a 1.2-kb band amplified with one primer. One locus influencing cytosine methylation at this restriction site was identified through genetic analysis of a BC1 population between C. grandis and P. trifoliata and was mapped to linkage group IV using an already developed core map. This technique for detecting methylation and methylation polymorphisms is simple and should be applicable to any eukaryotic species and to many situations where it is desirable to determine whether a sequence is methylated.

Characterization of the Citrus genome through analysis of restriction fragment length polymorphisms.

Studies on the nature of restriction fragment length polymorphisms (RFLPs) were undertaken to characterize the Citrus genome. This type of analysis has not been carried out with any other perennial crop. Citrus reticulata Blanco cv Clementine, C. xparadisi Macf. cv Duncan, and an F1 hybrid (LB 1-21) were used to determine what probe/enzyme combinations revealed polymorphisms in Southern analysis, and a backcross family (LB 1-21x'Clementine') of 65 randomly selected hybrid seedlings was used for some analyses. A majority (73%) of the clones examined from a PstI genomic library appeared to detect single-copy sequences based on RFLP banding patterns, while clones from a cDNA library revealed a lower percentage of single copy sequences. When hybridization stringencies were lowered, 21% of the genomic clones examined revealed greater copy numbers. PstI digestion of 'Duncan' DNA indicated abundant methylation, so the relatively high frequency of multiple-copy sequences observed at moderate stringency cannot be attributed to a lack of methylation of the Citrus DNA. The polymorphisms in banding patterns observed primarily resulted from insertions and/or deletions rather than from base substitutions, and a model is presented to account for the varying patterns obtained from individual probes with different restriction enzymes. Finally, a model for transposon activity in Citrus is proposed, based on observations made during the course of these studies.

An unusual group 2 LEA gene family in citrus responsive to low temperature.

Six cDNAs representing unique cold-induced sequences have been cloned from the hardy citrus relative Poncirus trifoliata. Among these, pBCORc115 and pBCORc119 were found to belong to the same gene family. Sequencing data indicated that pBCORc115 and pBCORc119 each contained an open reading frame, coding for a 19.8 kDa protein (COR19) and a smaller 11.4 kDa protein (COR11) respectively. Inspection of the deduced amino acid sequences revealed three large repeats in COR19, but only one was present in the COR11. Two elements: a Q-clustered tract and a K-rich motif were identified in each repeat. The K-rich motifs were similar to those of cotton D-11 and Group 2 LEA proteins. A Serine-cluster, a common feature in many Group 2 LEA-like proteins, was also found in these proteins, but it was in an unusual position at the carboxy-terminus. A bipartite motif of basic residues, similar to known nuclear targeting sequences, was also present in COR19 and COR11, suggesting that members of this protein family may have a nuclear targeting function. The expression of COR19 mRNA in response to cold acclimation, drought, flooding, and salinization was examined. COR19 expression in leaf tissue was induced in response to cold acclimation, but repressed during drought and flooding stress.

Cytotoxic effects of postharvest fungicides, ortho-phenylphenol, thiabendazole and imazalil, on isolated rat hepatocytes.

The cytotoxic effects of ortho-phenylphenol (OPP), imazalil (IMZ) and thiabendazole (TBZ) on isolated rat hepatocytes were investigated. Addition of IMZ and OPP to hepatocyte suspensions at a concentration of 0.75 mM resulted in acute cell death, accompanied by depletion of intracellular levels of glutathione and protein thiols. Both compounds rapidly depleted cellular ATP which consistently preceded the cell death. In addition, the cell death caused by IMZ was accompanied by the accumulation of intracellular malondialdehyde, indicating initiation of lipid peroxidation. During a 3-hr incubation period, TBZ did not affect these parameters. In mitochondria isolated from rat liver, IMZ and OPP impaired respiration related to oxidative phosphorylation. Based on these results, the order of toxic potency is IMZ > OPP > TBZ.

Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants.

Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T1 progeny contained T-DNA. Molecular characterization of one primary transformant and its T1 and T2 progeny plants established that T-DNA was integrated into the host genome.

Effect of captopril on functional mitral regurgitation in dilated heart failure: a randomised double blind placebo controlled trial.

OBJECTIVE: To determine the efficacy and dose requirements of captopril to reduce functional mitral regurgitation in patients with dilated heart failure. DESIGN: A randomised double blind placebo controlled parallel arm trial. Incremental daily doses of 25 mg, 50 mg and 100 mg captopril used for a four week period each for a total of 12 weeks preceded by a two week placebo washout. Twenty eight ambulatory patients (mean age 72) New York Heart Association (NYHA) class II or III with apparently controlled ischaemic dilated heart failure (ejection fraction 29% (0.04%)) on digoxin, diuretics, and nitrates were randomised. All had at least grade 2/4 functional mitral regurgitation (> 5 cm2 regurgitant area on colour flow Doppler). RESULTS: Twenty three patients completed the study (13 on placebo and 10 on captopril). Significant improvements were confined to the captopril group. Compared with placebo the following improvements were noted in the captopril treated group: mitral regurgitant area decreased from a threshold at 50 mg/day (p < 0.05, mean (95% confidence interval (95% CI)) 3.1 (0.2 to 6.0) cm2), with a further decrease at 100 mg/day (p < 0.01, mean (95% CI) 5.3 (3.1 to 7.5) cm2). Significant improvements in all the other measurements were noted only after 100 mg/day. Stroke volume increased (p < 0.01, mean (95% CI) 11, (1.4 to 21) ml), systemic vascular resistance decreased (p < 0.05, mean (95% CI) 414 (35 to 793) dyn s cm5), left atrial area decreased (p < 0.05, mean (95% CI) 4.3 (0.03 to 8.6) cm2), and deceleration time increased (p < 0.01, mean (95% CI) 52 ms (7 to 98) ms). Left ventricular diameter decreased marginally (p = 0.06, mean (95% CI) 4 (-0.05 to 9 mm). Duke activity index score increased (p < 0.001, median (95% CI) 6.8 (4.5 to 12) points). Heart rate, mean arterial blood pressure, serum creatinine, and serum potassium did not change with either placebo or captopril. No patient was withdrawn directly due to the side effects of captopril. In an open phase nine placebo patients given captopril in rapid increments reaching 100 mg/day in the fourth week showed similar improvements. CONCLUSION: Captopril is efficacious in reducing functional mitral regurgitation in dilated heart failure. Patients require and must tolerate high doses (50-100 mg/day) for additive effects over supervised conventional treatment to occur.

Effect of mitochondrial protein concentration on the efficiency of outer membrane removal by the cholesterol-selective detergent digitonin.

The effects of different mitochondrial protein concentrations on the efficiency of digitonin titration of the outer mitochondrial membrane were investigated in isolated rat liver mitochondria. Isolated mitochondria were subjected to treatment with digitonin concentrations ranging from 0 to 0.40 mg digitonin per mg protein. This digitonin concentration range was used in incubations containing 5 to 50 mg mitochondrial protein per ml. Significant differences in the efficiency of outer membrane removal by digitonin titration were noted at protein concentrations of less than 20 mg per ml. Estimation of the effective concentration of digitonin required to remove 50% of the outer membrane indicated that in general, as the protein concentration decreases, the amount of digitonin required to remove the outer membrane increases. Significant differences were also noted in the amount of digitonin needed for removal of 95% of the outer membrane between 5, 10 and 20 mg/ml with the effect of protein concentration disappearing above 20 mg/ml. No effect of protein concentration was found on the disruption of the inner membrane by digitonin as judged by leakage of matrix marker enzyme activity and by release of inner membrane marker enzyme activity. The conclusions of these studies indicate that at relatively low mitochondrial protein concentrations (< 20 mg/ml), the efficiency of digitonin in removing the outer membrane is substantially reduced.

Glucagonoma syndrome with increased lactate dehydrogenase isoenzymes: octreotide treatment.

Glucagonoma Syndrome is a rare syndrome comprising hyperglucagonemia, diabetes mellitus, necrolytic migratory erythema and hypoaminoacidemia in the setting of a glucagon producing, alpha cell tumour of the pancreas. We report a case of Glucagonoma Syndrome palliatively treated successfully with octreotide. In addition to classical clinical and biochemical findings, this patient also had a Glomus Jugulare tumour, and Empty Sella Syndrome and demonstrated an unusual pattern of plasma lactate dehydrogenase isoenzymes, features not previously reported in this syndrome.

The ECITTS integrated toxicity testing scheme: The application of in vitro test systems to the hazard assessment of chemicals.

A multicentre project has been designed, with the purpose of developing the concept of integrated in vitro testing. The aim is to use non-animal methods to assess the toxicological properties of chemicals, and to improve this assessment through the use of knowledge of mechanisms of toxic action. A number of tests or test batteries were selected within eight areas: basal cytotoxicity, irritancy, developmental toxicology, hepatotoxicity, nephrotoxicity, immunotoxicity, neurotoxicity and biokinetics. For each area, a number of calibration chemicals were selected. In an initial pilot study, the neurotoxic properties of five chemicals will be studied in combination with a biokinetic analysis, in which blood and brain concentrations will be predicted from biokinetic modelling.

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci.

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC1 progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70-80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.

Ascorbic acid uptake by isolated rat hepatocytes. Stimulatory effect of diquat, a redox cycling compound.

The toxicity of redox cycling compounds which generate the formation of active oxygen species is commonly accepted to be associated with a decrease of cellular reductants involved in cellular defence. However, when hepatocytes were incubated with diquat, an established redox cycler, in the presence of ascorbic acid (AA) (1 mM), the intracellular level of AA was increased. The effects of diquat on AA uptake were investigated in isolated rat hepatocytes. Incubation of hepatocytes with diquat plus AA (1 mM) resulted in about a 2-fold increased accumulation which occurred in a time-dependent manner reaching a steady state after 15 min at 37 degrees. The initial AA uptake rate was dependent on the AA concentration added. This process is described by Michaelis-Menten kinetics (apparent Km = 953 +/- 59 microM and Vmax = 2.68 nmol/min/10(6) cells). Characterization of AA accumulation showed it to be inhibited: by incubation at 4 degrees; with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an inhibitor of intracellular ATP production; by decreasing the extracellular Na+ concentration or incubating with ouabain; with pfloretin, a glucose transport inhibitor; and with glucose, a competitive inhibitor of AA transport. Replacement of AA with its oxidized form, dehydroascorbic acid, in the absence of diquat enhanced AA accumulation by 2.5-fold and apparently prevented further accumulation by added diquat. In addition, maintaining AA reduced with dithiothreitol inhibited the diquat effect. Diquat-induced AA accumulation was inhibited (65%) by desferrioxamine, a free-iron chelator, but not by catalase and/or superoxide dismutase or different antioxidants. In contrast, incubation with other active oxygen species generating systems including bipyridilium structural analogues, paraquat and benzyl viologen, had no effect on AA accumulation in hepatocytes. These results suggest that diquat-induced AA accumulation by hepatocytes occurs by a specific mediated transport system rather than as a consequence of cytotoxicity and may involve the presence of free-iron.