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Biomedical subjects

R J Roberts

Publications and source records attributed to R J Roberts.

At least 19 recordsLinked to original sources
PubMed1992

Environmental exposure to lead and children's intelligence at the age of seven years. The Port Pirie Cohort Study.

BACKGROUND: Exposure to lead in early childhood is thought to result in delayed neuropsychological development. As yet there is little longitudinal evidence to establish whether these effects persist into later childhood. METHODS: We measured IQ scores in 494 seven-year-old children from the lead-smelting community of Port Pirie, Australia, in whom developmental deficits associated with elevated blood lead concentrations had already been reported at the ages of two and four years. Exposure to lead was estimated from the lead concentrations in maternal blood samples drawn antenatally and at delivery and from blood samples drawn from the children at birth (umbilical-cord blood), at the ages of 6 and 15 months and 2 years, and annually thereafter. Data relating to known covariates of child development were collected systematically for each child throughout the first seven years of life. RESULTS: We found inverse relations between IQ at the age of seven years and both antenatal and postnatal blood lead concentrations. After adjustment by multiple regression for sex, parents' level of education, maternal age at delivery, parents' smoking status, socioeconomic status, quality of the home environment, maternal IQ, birth weight, birth order, feeding method (breast, bottle, or both), duration of breast-feeding, and whether the child's natural parents were living together, the relation with lead exposure was still evident for postnatal blood samples, particularly within the age range of 15 months to 4 years. For an increase in blood lead concentration from 10 micrograms per deciliter (0.48 mumol per liter) to 30 micrograms per deciliter (1.45 mumol per liter), expressed as the average of the concentrations at 15 months and 2, 3, and 4 years, the estimated reduction in the IQ of the children was in the range of 4.4 points (95 percent confidence interval, 2.2 to 6.6) to 5.3 points (95 percent confidence interval, 2.8 to 7.8). This reduction represents an approximate deficit in IQ of 4 to 5 percent. CONCLUSIONS: Low-level exposure to lead during early childhood is inversely associated with neuropsychological development through the first seven years of life.

Australia↗
PubMed1992

How M.MspI and M.HpaII decide which base to methylate.

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.

Base Sequence↗
PubMed1992

Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex.

The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.

Crystallization↗
PubMed1992

Trace quantitation of 4-hydroxy-2-nonenal in biological samples as its oxime-bis-tert.-butyldimethylsilyl derivative using 3-hydroxynonanal as an internal standard.

A gas chromatographic-mass spectrometric method for the determination of the lipid aldehyde 4-hydroxy-2-nonenal (4HNE) in trace quantities is described. The method utilizes the reaction of aldehydes with hydroxylamine leading to the formation of the oxime derivative. The aldehydes are recovered by octadecylsilyl solid-phase extraction and converted to the bis-tert.-butyldimethylsilyl derivatives for analysis using electron ionization. A novel 4HNE analogue, 3-hydroxynonanal, has been synthesized and is used as an internal standard. A limit of detection of approximately 1 pmol of 4 HNE in preparations of approximately 2.10(6) cells or 0.5 ml of whole blood, plasma or serum was observed. Standard addition analysis indicates that the method is accurate at these levels. Replicate analysis of the National Institutes of Standards and Technology Standard Reference Material SRM 909 indicates an average in-run precision of 8.1% and a between-run precision of 13.5% at an average concentration of 82.1 pmol/ml of reconstituted material.

Aldehydes↗
PubMed1992

Sequence-specific DNA binding by the MspI DNA methyltransferase.

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.

Bacteriophage lambda↗
PubMed1992

Finding errors in DNA sequences.

An algorithm is described that can detect certain errors within coding regions of DNA sequences. The algorithm is based on the idea that an insertion or deletion error within a coding sequence would interrupt the reading frame and cause the correct translation of a DNA sequence to require one or more frameshifts. If the coding sequence shows similarity to a known protein sequence then such errors can be detected by comparing the conceptual translations of DNA sequences in all six reading frames with every sequence in a protein sequence data base. We have incorporated these ideas into a computer program, called DETECT, that can serve as an aid to the experimentalist who is determining new DNA sequences so that obvious errors may be located and corrected. The program has been tested using raw experimental data and against sequences from the European Molecular Biology Laboratory data base, annotated as containing frameshifts. We have also tested it using unidentified open reading frames that flank known, annotated genes in the GenBank data base. Many potential errors are apparent and in some cases functions can be suggested for the "corrected" versions of these reading frames leading to the identification of new genes. As more sequences are determined the power of this method will increase substantially.

Adenylyl Cyclases↗
PubMed1992

Purification and characterization of the MspI DNA methyltransferase cloned and overexpressed in E. coli.

The MspI restriction-modification system, which recognizes the sequence 5'-CCGG-3', has been previously cloned and sequenced (1). We subcloned the methyltransferase gene (M.MspI) downstream of the ptac promoter in the multicopy vector pUC119 and overexpressed it in E. coli. Upon induction with IPTG, M.MspI constitutes more than 10% of cellular protein. A scheme has been devised to purify large amounts of biologically active M.MspI to apparent homogeneity from these overexpressing E. coli cells. Approximately 0.8 mg of pure M.MspI per gram of cells (wet weight) can be obtained. The apparent molecular weight of M.MspI is 49 kD, by SDS gel electrophoresis and 48-54 kD by gel filtration. At low concentrations (less than 0.4 mg/ml), the methyltransferase is a monomer in solution but at higher concentrations (greater than 3.0 mg/ml) it exists predominantly as a dimer. Polyclonal antibodies raised against M.MspI cross-react with the DNA-methyltransferases of several other restriction-modification systems.

Blotting, Western↗
PubMed1992

Mechanisms of cellular resistance to hydrogen peroxide, hyperoxia, and 4-hydroxy-2-nonenal toxicity: the significance of increased catalase activity in H2O2-resistant fibroblasts.

An H2O2-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line which demonstrates a 20-fold increase in catalase activity was utilized in the study of mechanisms responsible for cellular resistance to hydrogen peroxide, oxygen, and 4-hydroxy-2-nonenal toxicity. HA1 and OC14 cells were treated with 9 mM aminotriazole which resulted in a 60 to 80% reduction in catalase activity. Pretreatment with aminotriazole resulted in significant sensitization to the toxicity of 1-h exposures to exogenously applied H2O2, which was proportional to the reduction in catalase activity. Treatment with aminotriazole produced significant sensitization to the toxicity of 95% O2 after 45 h of O2 exposure but no sensitization to the toxicity of a 1-h exposure to 50 microM 4-hydroxy-2-nonenal. Inhibition of catalase activity by aminotriazole had no effect on the metabolism of 4-hydroxy-2-nonenal by either cell line tested. These results support the conclusion that in H2O2-resistant cells, catalase activity is a major determinant of cellular resistance to H2O2 toxicity, whereas catalase activity has a limited role in cellular resistance to an acute exposure to 95% O2 and is unrelated to cellular resistance to 4-hydroxy-2-nonenal.

Aldehydes↗
PubMed1992

The phenomenology of multiple partial seizure-like symptoms without stereotyped spells: an epilepsy spectrum disorder?

Consistent with previous reports by neuropsychiatrists, the results of the present investigation confirmed the existence of a neurobehavioral disorder characterized by the subjective experiencing of multiple cognitive, affective, and psychosensory phenomena similar to those associated with more classic partial seizure disorders. According to the literature, such patients typically respond favorably to anticonvulsants although they lack the customary motor manifestations of complex partial seizure (CPS) disorders and typically do not have stereotyped spells. This neuropsychiatric syndrome has recently been termed epilepsy spectrum disorder (ESD). In the present study, 30 patients with ESD were matched with equal numbers of treatment-refractory CPS patients and normal controls. All subjects were administered a standardized interview consisting of 35 cognitive, affective, and psychosensory partial seizure-like symptoms. The results indicated that ESD patients endorsed significantly more partial seizure-like symptoms than did CPS patients and controls. Relatively low levels of symptom endorsement by an unmatched psychiatric comparison sample indicated that the high levels of symptom endorsement by ESD patients could not be attributed to the presence of psychiatric dysfunction per se. Analysis of responding to 'foil' items unrelated to partial seizures indicated that high levels of symptom endorsement by ESD patients did not merely reflect a deviant response. Although ESD patients seldom present themselves at tertiary care epilepsy centers, the study of such patients is likely to be of relevance to mainstream epileptology.

Adult↗
PubMed1992

A stable O2-resistant cell line: role of lipid peroxidation byproducts in O2-mediated injury.

HA-1 hamster fibroblasts receiving fresh media every 24 h were continuously passaged in progressively increasing O2 concentrations for 18 mo (designated O2R95). These cells were significantly more resistant than parental HA-1 to clonogenic inactivation mediated by 95% O2 without media replacement. The O2R95 cell line exhibited increases in the activities of catalase (CAT), Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,Zn SOD), and glutathione peroxidase (GPx). O2R95 cells demonstrated uniformly distributed increased staining for CAT, MnSOD, Cu,Zn SOD, and GPx proteins, as determined by immunohistochemistry. Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a toxic byproduct of lipid peroxidation implicated in mechanisms of O2 toxicity, was examined in HA-1 and O2R95 cell lines. O2R95 cells were significantly more resistant to 4HNE cytotoxicity, which was accompanied by a significant increase in 4HNE metabolism. O2R95 cells also demonstrated an increase in total glutathione (GSH) and glutathione S-transferase (GST) activity, an enzymatic system believed to be involved with 4HNE metabolism. Furthermore, homogenates from O2R95 cells consumed greater quantities of 4HNE in the presence of NADPH (but not NADH, NAD+, or NADP+), suggesting that an enzyme(s) utilizing NADPH contributes to 4HNE metabolism, resistance to 95% O2 and 4HNE as well as increased total GSH, antioxidant enzyme activities, and NADPH-dependent metabolism of 4HNE, persisted in O2R95 cells for 75 days of growth in 21% O2. These findings are compatible with the hypothesis that aldehydic byproducts of lipid peroxidation contribute to mechanisms of O2 toxicity and the selective pressure exerted by exposure of cells to hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The effect of monosaturated and polyunsaturated fatty acids on oxygen toxicity in cultured cells.

The influence of oleic, linoleic (LIN), and eicosapentaenoic (EPA) acids incorporated into cellular lipids on susceptibility to O2-induced toxicity was evaluated in Chinese hamster fibroblasts (HA1) using a clonogenic cell survival assay. Fatty acid incorporation was achieved by incubating HA1 cells in 21% O2 for 72 h in the presence or absence of media supplemented with 25 microM oleic acid, 25 microM LIN, or 2, 4, and 25 microM EPA. This fatty acid incorporation period increased the percentage of composition in phospholipids 2-fold for oleic acid, 6-fold for LIN, and 6- to 20-fold for EPA. Vitamin E, total glutathione, superoxide dismutase activity, glutathione transferase activity, and catalase activity were unchanged, relative to control, in the 25-microM EPA-treated group, and only total glutathione was elevated in the LIN-treated group. After the incorporation period, the cells were placed in non-fatty acid supplemented media and exposed to 95% O2, and clonogenic survival responses were evaluated at time intervals up to 100 h. Sensitization to O2 toxicity in EPA-treated cells was apparent after 24 h of O2 exposure, whereas LIN-treated cells were significantly (p less than 0.05) sensitized to hyperoxia after 54 h of exposure, indicating that EPA was a more potent sensitizer for O2 injury. Furthermore, cells supplemented with 4 and 25 microM EPA were more sensitive to O2 toxicity than cells supplemented with 2 microM EPA. In contrast, cells treated with 25 microM oleic acid were significantly more resistant to O2 toxicity at 51, 72, and 98 h of O2 exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The sequence specificity domain of cytosine-C5 methylases.

Prokaryotic DNA[cytosine-C5]methyltransferases (m5C-methylases) share a common architectural arrangement of ten conserved sequence motifs. A series of eleven hybrids have been constructed between the HpaII (recognition sequence: Cm5CGG) and HhaI (recognition sequence: Gm5CGC) DNA-methylases. The hybrids were over-expressed in E.coli and their in vivo methylation phenotypes investigated. Six were inactive by our assay while five of them retained partial methylation activity and full specificity. In all five cases the specificity matched that of the parent methylase which contributed the so-called variable region, located between conserved motifs VIII and IX. This was the only sequence held in common between the active hybrids and for the first time provides unequivocal evidence that the specificity determinants of the mono-specific m5C-methylases are located within the variable region. Correlation of the hybrid methylase structure with the efficiency of methylation suggests that conserved motif IX may interact with the variable region whereas motif X most probably interacts with the N-terminal half of the molecule.

Amino Acid Sequence↗
PubMed1991

Replacement of media in cell culture alters oxygen toxicity: possible role of lipid aldehydes and glutathione transferase in oxygen toxicity.

Replacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity. Following 100 hr of exposure to 95% or 80% O2, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 x 10(-3) when the culture media was replaced only at the onset of the O2 exposure. Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 x 10(-1) (95% O2) and 1.9 x 10(-1) (80% O2) at 95 hr. Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O2 for 144 hr with media replacement. These O2-exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 microM 4HNE to reach 30% survival as compared to density-matched normoxia control. Furthermore, during 40 and 60 min of exposure to 4HNE, the O2-preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control. The activity of glutathione S-transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O2-preexposed cells compared with controls. Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper-zinc superoxide dismutase activities at the time of 4HNE treatment in the O2-preexposed cells relative to density-matched control. The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O2 exposure. Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST-catalyzed detoxification. These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidation contribute to hyperoxic injury.

Aldehydes↗
PubMed1991

Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal.

The involvement of glutathione (GSH) dependent processes in the detoxification of 4-hydroxy-2-nonenal (4HNE) was investigated using Chinese hamster fibroblasts and clonogenic cell survival. GSH reacted, in a dose-dependent fashion, with 4HNE in phosphate buffer at pH 6.5, leading to the disappearance of 4HNE. The addition of glutathione transferase activity (GST) facilitated a more rapid disappearance of 4HNE but the reaction was still dependent on the concentration of GSH. When cell cultures were exposed to the reaction mixtures, 4HNE cytotoxicity was also reduced in a manner which was dependent on the concentration of GSH. When 2.16- or 1.08-mM GSH were incubated in phosphate buffer with 1.08-mM 4HNE in the presence or absence of GST, then mixed with media and placed on cells for 1 h, the cytotoxicity associated with exogenous exposure to free 4HNE was abolished. GSH depletion (greater than 90%) using buthionine sulfoximine (BSO) was accomplished in control (HA1) and H2O2-resistant variants derived from HA1. GSH depletion resulted in enhanced cytotoxicity of 4HNE in all cell lines. This BSO-induced sensitization to 4HNE cytotoxicity was accompanied by a significant reduction in the ability of cells to metabolize 4HNE. The magnitude of the sensitization to 4HNE toxicity caused by GSH depletion was similar to the magnitude of the reduction in the ability of cells to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for protection against aldehydic by-products of lipid peroxidation.

Aldehydes↗
PubMed1991

Dichotic listening failure in dysphoric neuropsychiatric patients who endorse multiple seizure-like symptoms.

In the present investigation, the dichotic word listening performance of a sample of 25 dysphoric neuropsychiatric patients who endorsed multiple partial seizure-like symptoms was compared with that of matched samples of normal controls and patients with mood disorders who did not endorse multiple seizure-like symptoms. Eighty percent of the patients who endorsed multiple episodic phenomena failed the dichotic listening task, compared with 8% of normal controls and 28% of patients with typical mood disorders. After treatment with carbamazepine, a subsample of polysymptomatic patients manifested significantly fewer seizure-like symptoms. This clinical improvement was typically associated with markedly improved dichotic listening performance in most cases. The results are consistent with our previous hypothesis that "subclinical" electrophysiological dysfunction may severely disrupt the normal transmission and processing of auditory information. Because it is sensitive to this type of presumed cerebral dysfunction and relatively specific, impaired dichotic listening performance is likely to be a useful clinical marker for this complex neuropsychiatric syndrome.

Adult↗
PubMed1991

Spontaneously productive C-type retrovirus infection of fish cell lines.

The spontaneous production and release of morphologically typical, 85 to 90 nm diameter C-type retrovirus particles from four cell lines derived from three species of warmwater fish have been identified. Virus pellets from cell culture supernatants showed high levels of Mn(2+)-dependent reverse transcriptase activity at 24 degrees C. Peak enzyme activity was associated with a 1 x 16 g/ml sucrose gradient fraction. All four isolates induced a cytolytic infection of a bluegill fry cell line within 6 to 10 days.

Animals↗