Vaccine use and abuse: a practitioner's viewpoint.
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Animals↗
Biomedical subjects
G A Moore
Publications and source records attributed to G A Moore.
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.
Blotting, Southern↗
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.
Citrus↗
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.
Acoustic Stimulation↗
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.
Adult↗
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.
Amino Acid Sequence↗
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.
Adult↗
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.
Adenosine Triphosphate↗
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.
Base Sequence↗
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.
Adaptation, Physiological↗
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.
Animals↗
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.
Animals↗
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.
Empty Sella Syndrome↗
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.
Aged↗
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.
Animals↗
Accessibility of hepatocyte protein thiols to monobromobimane.
The amino-acid residue specificity of monobromobimane (mBBr) and its accessibility to cellular protein cysteine residues were investigated. mBBr reacted selectively with the sulfhydryl group of both the free amino acid cysteine and bovine serum albumin. Incubation of isolated hepatocytes with mBBr resulted in a concentration-dependent formation of protein-bound mBBr fluorescence in the cytosolic, mitochondrial and microsomal fractions, which was not fully saturated with up to 16 mM mBBr. SDS-PAGE resolution of the proteins revealed that the major portion of increased protein-bound mBBr fluorescence that occurred at high mBBr concentrations was due to covalent binding to proteins. A minor portion (10-16% in the microsomal fraction) of protein-bound mBBr fluorescence was removed by SDS-PAGE and is therefore concluded to be due to physical entrapment of fluorescent mBBr reaction products. The accessibility of mBBr, assayed as the degree of depletion of total protein cysteine residues, was similar to N-ethylmaleimide (NEM) in isolated microsomes. By contrast, in the cytosol a markedly lower amount of protein cysteine residues were labelled by mBBr as compared to NEM. In both organelle fractions p-BQ was the most efficient thiol-depleting reagent. It is concluded that mBBr is a suitable reagent for the analysis of the cellular protein thiol status and of its xenobiotic-induced alterations when used at high concentrations; however, it should be considered that, (i) the relative accessibility of mBBr and a particular xenobiotic to cellular protein thiol residues may be different, and (ii) physically entrapped fluorescent reaction products of mBBr should be removed when quantitating protein thiol levels.
Amino Acids↗
Submitochondrial localization of the NAD+ glycohydrolase. Implications for the role of pyridine nucleotide hydrolysis in mitochondrial calcium fluxes.
The submitochondrial location of the NAD+ glycohydrolase (NADase) and its role in mitochondrial ADP-ribosyl transfer reactions were investigated in isolated rat liver mitochondria. The NADase catalyzes the hydrolysis of NAD+ to ADP-ribose and nicotinamide. Hydrolysis of intramitochondrial NAD+ has been suggested to be the first step in a nonenzymatic mono(ADP-ribosylation) by free ADP-ribose of an inner membrane-associated acceptor peptide and that this protein is involved in regulating calcium efflux from mitochondria during exposure to oxidants. The results of the present study indicate that mitochondrial NADase activity lies outside the matrix space, however. This was determined by assessing the rates of hydrolysis of externally added NAD+ by intact mitochondria and comparing these rates with those obtained when the mitochondria were permeabilized with detergent. No significant difference was observed in the rate of NAD+ hydrolysis when detergent was added indicating that NAD+ hydrolysis by mitochondria does not necessitate its access to the matrix space. The submitochondrial location of the NADase was investigated further by digitonin titration of isolated mitochondria. Digitonin titration of the mitochondria released NADase activity with the outer membrane marker, monoamine oxidase. The digitonin titration data also suggest that the outer membrane is the exclusive location of the NADase. The incorporation of radioactive label derived from [3H]NAD+ into submitochondrial particles proceeds at comparable rates in the absence or presence of NADase activity, indicating that the production of free ADP-ribose is not necessary for intramitochondrial ADP-ribosyl transfer reactions. Thus the conclusions of this study suggest that due to the submitochondrial location of the NADase, it does not participate in intramitochondrial pyridine nucleotide hydrolysis or nonenzymatic mono(ADP-ribosylation) during oxidative stress in mitochondria.
Adenosine Diphosphate Ribose↗
Relationship between metabolism and cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.
The relationship between the metabolism and the cytotoxicity of ortho-phenylphenol (OPP) was investigated using isolated rat hepatocytes. Addition of OPP (0.5-1.0 mM) to the hepatocytes caused a dose-dependent toxicity; 1.0 mM OPP caused acute cell death. Pretreatment of hepatocytes with SKF-525A (50 microM, a non-toxic level) enhanced the cytotoxicity of OPP (0.5-1.0 mM). This was accompanied by inhibition of OPP metabolism. Conversely, OPP at low concentrations (0.5 or 0.75 mM) was converted sequentially to phenyl-hydroquinol (PHQ) and then to glutathione (GSH) conjugate in the cells. The concentrations of both metabolites, especially PHQ-GSH conjugate, were very low in hepatocytes exposed to 1.0 mM OPP alone as well as with SKF-525A. The cytotoxicity induced by 0.5 mM OPP was enhanced by the addition of diethylmaleate (1.25 mM) which continuously depletes cellular GSH. In contrast, additions to hepatocytes of 5 mM of dithiothreitol, cysteine, N-acetyl-L-cysteine or ascorbic acid significantly inhibited the cytotoxicity induced by 0.5 mM PHQ; GSH, protein thiols and ATP losses were also prevented. Further, these compounds depressed the rate of PHQ loss in hepatocyte suspensions. These results indicate that the acute cytotoxicity caused by the high dose (1.0 mM) of OPP is associated with direct action by the parent compound; at low doses (0.5-0.75 mM) of OPP, the prolonged depletion of GSH in hepatocytes enhances the cytotoxicity induced by PHQ.
Adenosine Triphosphate↗