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Biomedical subjects

R A Jones

Publications and source records attributed to R A Jones.

At least 19 recordsLinked to original sources
PubMed1999

Liver poly(ADP-ribose)polymerase is resistant to cleavage by caspases.

In hepatocytes the DNA repair enzyme poly(ADP-ribose)polymerase (PARP) is not proteolytically cleaved during apoptosis. The reason for this was investigated using a cell-free system that consisted of isolated nuclei from hepatocytes or thymocytes and cytosolic extracts from hepatocytes or thymocytes undergoing apoptosis. It was found that liver PARP is resistant to proteolytic cleavage by the caspases present in the cytosolic extracts. Furthermore, liver PARP was not cleaved by recombinant human caspase-3. It is concluded that PARP proteolysis cannot be used as a marker for hepatocyte apoptosis.

Animals↗
PubMed1999

Use of the mean transit time of an intravascular contrast agent as an exchange-insensitive index of myocardial perfusion.

A simple two-compartment model was used to study the effects of water exchange on the signal produced by an inversion recovery prepared rapid gradient-echo sequence during the first passage of a low dose of an intravascular contrast agent. Water exchange at intermediate rates of exchange (1-10 Hz) between the vascular and extravascular spaces caused the form of the signal changes during the first pass to be dependent on both the fractional sizes of the vascular and extravascular compartments and on the exchange rate. Unless the effects of exchange are minimized by using a very short inversion time, parameters such as the peak height and area under the curve will be affected by regional and/or pathological variations in the exchange rate and the size of the vascular fraction. The mean transit time (MTT) is, however, less affected by water exchange. Experimental first-pass data produced by intravascular low-dose injections of iron oxide particles were studied in five pigs at 0.5 T. The MTT as derived from the first-pass curves, without deconvolution with the arterial input function, was well correlated with the myocardial blood flow (MBF) as measured using radioactive microspheres (r = 0.70, n = 52, P < 0.01). Other first-pass parameters such as the peak height or area under the curve exhibited either a poorer, or no, correlation with the MBF. The data suggest that the MTT of the first pass of an intravascular contrast agent may be a robust, quantitative method for assessing myocardial blood flow in patients.

Animals↗
PubMed1999

Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium.

A hyperthermophilic and amylolytic prokaryote, designated Rt3, was isolated from a thermal spring near Rotorua, New Zealand. The 16S rRNA gene of Rt3 was cloned and sequenced with the aim of determining its phylogenetic affiliations. The phylogenetic analysis of this sequence, which included a selection of archaebacterial and eubacterial 16S rRNA sequences, indicates that Rt3 most likely belongs to the archaebacterial order Thermococcales. An amylase gene (amyA) from Rt3, encoding a highly thermostable amylase activity, was cloned and its DNA sequence determined. Transcriptional signals typical of archaebacteria were evident in this sequence. The sequence is homologous to a broad range of enzymes from the AMY superfamily and contains a typical N-terminal signal peptide. Phylogenetic analysis and comparison of structural features with other AMY superfamily enzymes reveals that, firstly, the closest homologues of the Rt3 amylase are members of the Bacillus and Plant alpha-amylase groups; and secondly, that the Rt3 amylase is closely related to only one other currently known archaebacterial enzyme, i.e. an (AMY superfamily) alpha-amylase from Natronococcus.

Amino Acid Sequence↗
PubMed1998

Solution structure of a Na cation stabilized DNA quadruplex containing G.G.G.G and G.C.G.C tetrads formed by G-G-G-C repeats observed in adeno-associated viral DNA.

We have applied NMR and molecular dynamics computations including intensity based refinement to define the structure of the d(G-G-G-C-T4-G-G-G-C) dodecanucleotide in 100 mM NaCl solution. The G-G-G-C sequence is of interest since it has been found as tandem repeats in the DNA sequence of human chromosome 19. The same G-G-G-C sequence is also seen as islands in adeno-associated virus, a human parvovirus, which is unique amongst eukaryotic DNA viruses in its ability to integrate site-specifically into a defined region of human chromosome 19. The d(G-G-G-C-T4-G-G-G-C) sequence forms a quadruplex in Na cation containing solution through head-to-tail dimerization of two symmetry-related stem-hairpin loops with adjacent strands antiparallel to each other around the quadruplex. The connecting T4 loops are of the lateral type, resulting in a quadruplex structure containing two internal G.G.G.G tetrads flanked by G.C.G.C tetrads. The G(anti).G(syn).G(anti).G(syn) tetrads are formed through dimerization associated hydrogen bonding alignments of a pair of Hoogsteen G(anti).G(syn) mismatch pairs, while the G(anti).C(anti).G(anti).C(anti) tetrads are formed through dimerization associated bifurcated hydrogen bonding alignments involving the major groove edges of a pair of Watson-Crick G.C base-pairs. The quadruplex contains two distinct narrow and two symmetric wide grooves with extensive stacking between adjacent tetrad planes. The structure of the quadruplex contains internal cavities that can potentially accommodate Na cations positioned between adjacent tetrad planes. Three such Na cations have been modeled into the structure of the d(G-G-G-C-T4-G-G-G-C) quadruplex. Finally, we speculate on the potential role of quadruplex formation involving G.G.G.G and G.C.G.C tetrads during the integration of the adeno-associated parvovirus into its target on human chromosome 19, both of which involve stretches of G-G-G-C sequence elements.

Chromosomes, Human, Pair 19↗
PubMed1998

Transcriptional regulation of MHC class I gene expression in rat oligodendrocytes.

MHC class I molecules are normally expressed at very low levels in the brain and their up-regulation in response to cytokines and viral infections has been associated with a number of neurological disorders. Here we demonstrate that the down-regulation of surface class I molecules in differentiated primary rat oligodendrocytes was accompanied by reduced steady-state levels of class I heavy-chain mRNA. Transient expression assays were performed in oligodendrocytes and fibroblasts, using a mouse H-2Kb class I promoter chloramphenicol acetyltransferase plasmid termed pH2KCAT (which contained 5'-flanking sequences from -2033 to +5 bp of the H-2Kb gene relative to the transcriptional start site at +1 bp). These assays showed that H-2Kb promoter activity was reduced in oligodendrocytes but not in class I-expressing fibroblasts. H-2Kb promoter activity was up-regulated in oligodendrocytes co-transfected with a plasmid expression vector encoding the transcriptional activator tax of human T-cell leukaemia virus type I, showing that down-regulation of promoter activity was reversible. Deletion mutant analysis of the H-2Kb promoter revealed the presence of negative regulatory elements that were functional in oligodendrocytes at -1.61 to -1.07 kb and -242 to -190 bp. Deletion of sequences in pH2KCAT encompassing the downstream element totally abolished promoter activity in both oligodendrocytes and fibroblasts, whereas a deletion within the upstream negative regulatory element increased promoter activity specifically in oligodendrocytes. The upstream negative regulatory element also down-regulated a linked heterologous herpes simplex virus thymidine kinase promoter in oligodendrocytes, but not in fibroblasts. Gel retardation assays using overlapping DNA probes that spanned the entire -1.61 to -1.07 kb region revealed the presence of a number of DNA-binding activities that were present in oligodendrocyte, but not in fibroblast nuclear extracts.

Animals↗
PubMed1998

Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases.

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.

Animals↗
PubMed1998

Signal undershoots following visual stimulation: a comparison of gradient and spin-echo BOLD sequences.

Gradient-echo (GRE) and spin-echo (SE) EPI BOLD sequences were used to quantitate the effect of visual stimulation. Both sequences showed a positive BOLD response during stimulation and a negative BOLD response in the interstimulation intervals. The relaxation rate changes during stimulation were larger for the GRE sequence than for the SE sequence, whereas in the interstimulation intervals they were not significantly different. In both cases, the ratio of the GRE/SE relaxation rate changes were consistent with BOLD effects in larger vessels despite the well-known lower sensitivity of the SE sequence to the extravascular component of the BOLD effect in larger vessels. The most probable explanation of this result is that a significant fraction of the observed changes originated from the intravascular component of the BOLD effect. The SE sequence depicted smaller areas of activation than the GRE sequence with more than 85% of the pixels being depicted as significant by the SE sequence being also significant in the GRE activation maps. However, for the reverse comparison, an overlap of only 35% was observed, with many of the strongly correlated GRE pixels showing weak correlations in the corresponding SE activation image. Our results, together with the fact that signal undershoots have not been observed by groups using MR sequences that measure absolute flow changes for similar stimulation paradigms, suggest that the undershoot may be due to alterations in the blood volume and/or hematocrit during stimulation that normalize at a slower rate than the changes in blood flow after the cessation of the stimulation, leading to a poststimulation signal undershoot.

Adult↗
PubMed1998

Degenerative mitral regurgitation: when should we operate?

BACKGROUND: Left untreated, severe mitral regurgitation in asymptomatic patients can lead to irreversible cardiac damage, which can develop with little warning. Over the period of this study, we have tended to operate earlier in the disease process and on less symptomatic patients. We report here our experience. METHODS: Between January 1985 and June 1996, 710 patients with mitral regurgitation underwent operations. Three hundred twenty-nine (213 male and 116 female with a mean age of 65.5 years) had degenerative mitral valve disease and of this group, 169 patients underwent repair and 160, replacement. RESULTS: The overall operative mortality was 4 patients (1.2%). There were no operative deaths among patients having isolated mitral valve repair. Survival at 1 year, 5 years, and 10 years was 94%+/-1.4% (+/- the standard error of the mean), 77%+/-2.9%, and 41%+/-5.8%, respectively. Survival was significantly better in the group having repair (p < 0.05). Ten patients (6%) in the repair group and 13 (8%) in the replacement group required reoperation. Increased age, worse left ventricular function, type of operation (replacement worse than repair), and increased left ventricular size were significantly associated with poorer survival. CONCLUSIONS: These data confirm the superior results achieved with mitral valve repair and support early mitral valve repair before functional deterioration.

Adolescent↗
PubMed1998

A patient with carnitine-acylcarnitine translocase deficiency with a mild phenotype.

Carnitine-acylcarnitine translocase deficiency, a rare beta-oxidation defect, is manifest in most cases by cardiomyopathy and death in early childhood. We report an affected patient, 3 years of age, who has had no serious complications. The residual enzyme activity in fibroblasts was higher than in previously reported patients, which may explain the benign clinical course.

Blood Glucose↗
PubMed1998

Formation of an amino-acid-binding pocket through adaptive zippering-up of a large DNA hairpin loop.

BACKGROUND: In vitro selection has identified DNA aptamers that target cofactors, amino acids, peptides and proteins. Structure determination of such ligand-DNA aptamer complexes should elucidate the details of adaptive DNA structural transitions, binding-pocket architectures and ligand recognition. We have determined the solution structure of the complex of a DNA aptamer containing a guanine-rich 18-residue hairpin loop that binds L-argininamide with approximately 100 microM affinity. RESULTS: The DNA aptamer generates its L-argininamide-binding pocket by adaptive zippering up the 18-residue loop through formation of Watson-Crick pairs, mismatch pairs and base triples, while maximizing stacking interactions. Three of the four base triples involve minor-groove recognition through sheared G.A mismatch formation. The unique fold is also achieved through positioning of an adenine residue deep within the minor groove and through nestling of a smaller loop within the larger loop on complex formation. The accessibility to the unique L-argininamide-binding pocket is restricted by a base pair that bridges across one side of the major-groove-binding site. The guanidinium group of the bound L-argininamide aligns through intermolecular hydrogen-bond formation with the base edges of nonadjacent guanine and cytosine residues while being sandwiched between the planes of nonadjacent guanine residues. CONCLUSIONS: The available structures of L-arginine/L-argininamide bound to their DNA and RNA targets define the common principles and patterns associated with molecular recognition, as well as the diversity of intermolecular hydrogen-bonding alignments associated with the distinct binding pockets.

Amino Acids↗
PubMed1998

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments.

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100 degrees C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10(2) cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples.

Catechol 2,3-Dioxygenase↗
PubMed1998

Trisomy 12 is seen within a specific subtype of B-cell chronic lymphoproliferative disease affecting the peripheral blood/bone marrow and co-segregates with elevated expression of CD11a.

In order to delineate the specific morphological and immunophenotypic features of B-cell lymphoproliferative disorders associated with trisomy 12, 172 sequential unselected cases of CD19+CD5+ B-cell disorders, primarily affecting the peripheral blood and bone marrow, were studied. Trisomy 12 was found in 24 cases (13.9%), with all cases morphologically classified as either CLL-PL or CLL-mixed by FAB criteria. Trisomy 12 was not found in any cases of typical CLL. Trisomy 12 cases demonstrated a significant higher expression of CD11a (P<0.0001) and CD20 (P<0.0006) when compared to cases with the equivalent morphology and immunophenotype, but without the chromosomal abnormality. Trisomy 12 cases also demonstrated a higher frequency of FMC7, CD38 expression and moderate to strong surface immunoglobulin staining. However, no correlation was detected between the percentages of trisomy 12 cells and cells expressing CD11a, CD38, FMC7 or sIg mean fluorescent intensity. Cells from trisomy 12 positive cases were sorted according to their CD11a expression using fluorescent activated cell sorting. There was a significant increase in the percentage of trisomy 12 cells within the CD11a+ sorted fraction compared to the unsorted population (P < 0.05), implying that trisomy 12 is associated with increased expression of CD11a. With the highly specific morphological and immunophenotypic features demonstrated by trisomy 12 cases in this study, it is highly likely that these cases constitute a specific group of B-cell lymphoproliferative disorders.

Adult↗
PubMed1998

S100 beta increases levels of beta-amyloid precursor protein and its encoding mRNA in rat neuronal cultures.

S100beta has been implicated in the formation of dystrophic neurites, overexpressing beta-amyloid precursor protein (betaAPP), in the beta-amyloid plaques of Alzheimer's disease. We assessed the effects of S100beta on cell viability of, neurite outgrowth from, and betaAPP expression by neurons in primary cultures from fetal rat cortex. S100beta (1-10 ng/ml) enhanced neuronal viability (as assessed by increased mitrochondrial activity and decreased lactic acid dehydrogenase release) and promoted neurite outgrowth. Higher levels of S100beta (100 ng/ml, but not 1 microg/ml) produced qualitatively similar, but less marked, effects. S100beta also induced increased neuronal expression of the microtubule-associated protein MAP2, an effect that is consistent with trophic effects of S100beta on neurite outgrowth. S100beta (10 and 100 ng/ml) induced graded increases in neuronal expression of betaAPP and of betaAPP mRNA. These results support our previous suggestion that excessive expression of S100beta by activated, plaque-associated astrocytes in Alzheimer's disease contributes to the appearance of dystrophic neurites overexpressing betaAPP in diffuse amyloid deposits, and thus to the conversion of these deposits into the diagnostic neuritic beta-amyloid plaques.

Amyloid beta-Protein Precursor↗
PubMed1997

RNA synthesis using a universal, base-stable allyl linker.

The application of a universal allyl linker, 9-O-(4,4'-dimethoxytrityl)-10-undecenoic acid, to the solid phase synthesis of RNA molecules is described. Use of this linker simplifies significantly the isolation and purification steps in RNA synthesis. The linker is universal in that it does not contain a nucleoside. The 3'terminal nucleoside is instead attached to the support in the first coupling step. The resultant RNA fragment is then obtained as the 3'-phosphate. The linker is base-stable, and thus all reagents used during deprotection can simply be washed away, leaving the RNA attached. Further, tritylated short fragments resulting from chain cleavage for any reason are also washed away before separation from the support. This linker is compatible with any current synthetic methodology and any amino functionalized support. Of course, silica supports would not be compatible with fluoride reagents. It could also be used to advantage for other applications. Because it is cleaved under conditions orthogonal to those used during many common reactions, the range of post-synthetic manipulations that can be carried out without cleavage from the support is extended significantly.

Acetates↗
PubMed1997

A study of the contribution of changes in the cerebral blood volume to the haemodynamic response to anoxia in rat brain.

A susceptibility contrast agent which does not pass into the extra-cellular space was used to study the effect of changes in the relative cerebral blood volume (CBV) on the haemodynamic response to anoxia, for both normal and ischaemic brain tissue, in a rat model of acute focal ischaemia. In non-ischaemic tissue a strong CBV component was observed in the haemodynamic response, both during and after anoxia. During anoxia the change in the CBV of the non-ischaemic tissue was estimated to be 40% in the caudate putamen and 70% in the frontal-parietal cortex. For severely ischaemic tissue (ischaemic caudate putamen) there was no change in the CBV during anoxia while in areas of moderate ischaemia (ischaemic frontal parietal cortex) a change of 20% was observed. The effect of the contrast agent on spin-echo images was consistent with a small reduction in the microvascular blood volume of the ischaemic tissue.

Animals↗
PubMed1997

Exposure of nerve growth factor-treated PC12 rat pheochromocytoma cells to a modulated radiofrequency field at 836.55 MHz: effects on c-jun and c-fos expression.

Rat PC12 pheochromocytoma cells have been treated with nerve growth factor and then exposed to athermal levels of a packet-modulated radiofrequency field at 836.55 MHz. This signal was produced by a prototype time-domain multiple-access (TDMA) transmitter that conforms to the North American digital cellular telephone standard. Three slot average power densities were used: 0.09, 0.9, and 9 mW/cm2. Exposures were for 20, 40, and 60 min and included an intermittent exposure regimen (20 min on/20 min off), resulting in total incubation times of 20, 60, and 100 min, respectively. Concurrent controls were sham exposed. After extracting total cellular RNA, Northern blot analysis was used to assess the expression of the immediate early genes, c-fos and c-jun, in all cell populations. No change in c-fos transcript levels were detected after 20 min exposure at each field intensity (20 min was the only time period at which c-fos message could be detected consistently). Transcript levels for c-jun were altered only after 20 min exposure to 9 mW/cm2 (average 38% decrease).

Adrenal Gland Neoplasms↗