Reverse transcription of mRNA by Thermus aquaticus DNA polymerase followed by polymerase chain reaction amplification.
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Avian Myeloblastosis Virus↗
Biomedical subjects
M D Jones
Publications and source records attributed to M D Jones.
Complex nature of the major viral polyadenylated transcripts in Epstein-Barr virus-associated tumors.
The most abundant polyadenylated viral transcripts in the Epstein-Barr virus (EBV)-associated tumor nasopharyngeal carcinoma are a family (apparent sizes, 4.8, 5.2, 6.2, and 7.0 kb) of highly spliced cytoplasmic RNAs expressed from the BamHI-I and -A regions of the viral genome in an antisense direction with respect to several viral lytic functions encoded within the same region and concerned with the lytic cycle of the virus. We have called these complementary-strand transcripts. They are also expressed in B cells, including Burkitt's lymphoma and EBV-immortalized marmoset cell lines, and tumors generated in cottontop tamarins in response to EBV infection, but at a lower level. The complete structure of the major 4.8-kb RNAs (seven or eight exons) was determined in this study; the larger, but related, transcripts appear to be produced by differential splicing. The transcriptional promoter for the major complementary-strand transcripts, located in BamHI-I, contains several well-characterized transcriptional control elements (E2A, SP1, and AP1) and is functionally active in both B lymphocytes and epithelial cells. It appears to be a bifunctional viral promoter, as it also contains the initiation codon for a gene (BILF2) that encodes a glycoprotein that is expressed off the other strand. Splicing events create a number of small AUG-initiated open reading frames, one of which has homology to functionally significant regions of the EBV-encoded nuclear antigen 2 and to E2 (in papillomavirus). The complex nature of these transcripts and their potential role in the virus association with malignancy are considered.
Amino Acid Sequence↗
Pial microvascular hemodynamics in anemia.
Isovolemic hemodilution and subsequent anemia increase cerebral blood flow (CBF). We hypothesized that pial microvascular pressure also increases with hemodilution and that arteriolar diameter varies concurrently as a myogenic autoregulatory response. First- and second-order arterioles (31-92 microns, n = 29) and large venules (65-215 microns, n = 17) were studied in thiopental-anesthetized rats. Microvascular pressure was determined using the servo-null technique, and vessel diameters were obtained directly from a video monitoring system. We measured the increase in CBF (radiolabeled microspheres) that accompanies hemodilution in a separate group of animals (n = 20). Hematocrit was reduced to 16-36% with homologous plasma (hemodilution group, n = 13) or held constant with homologous whole blood (control group, n = 4). In control animals, arteriolar and venular diameter varied +/- 1-2 microns from baseline values, and microvascular pressure remained unchanged from baseline. In the hemodilution group, CBF increased, but there was no systematic pial vasodilation. Furthermore, intraluminal pressure did not increase in pial microvessels, suggesting that proximal vasodilation was negligible even at the lowest hematocrit studied. Vascular resistance fell proportionately in both large vessel and microvascular segments. We conclude that experimental anemia does not produce alterations in microvascular pressure in rats, and the hyperemia accompanying hemodilution is largely viscosity mediated.
Anemia↗
Fetal responses to acute maternal cocaine injection in sheep.
Maternal cocaine abuse has been associated with neonatal neurological and neurobehavioral problems of unknown pathogenesis. We administered a single intravenous dose of cocaine (2 mg/kg) to 12 unanesthetized pregnant sheep; their fetuses had been catheterized in utero 2 days before the study. We measured fetal cerebral blood flow (CBF), cerebral metabolic rate of O2 (CMRO2), mean arterial blood pressure (MAP), and blood gases before and 2, 5, 15, and 30 min after maternal cocaine injection. Fetal CBF increased by 37 +/- 33% (mean +/- SD) at 5 min and returned to baseline by 15 min. Regional brain blood flow changes paralleled CBF changes with the greatest increases occurring in cerebellum (54 +/- 43%) and brain stem (54 +/- 52%). Cerebral vascular resistance was decreased for cerebellum (22%) and brain stem (19%) but was unchanged for cerebral hemispheres and caudate. Increased CBF at 5 min was associated with a 20 +/- 9% increase in fetal MAP and a 38 +/- 13% decrease in fetal arterial O2 content. Fetal CMRO2 was unchanged. There was a decrease in fetal intestinal blood flow at 2 min, an increase in myocardial, adrenal, and renal blood flow at 5 min, and no change in placental blood flow. Maternal cocaine injection causes fetal hypoxemia, hypertension, and increased CBF. Possible mechanisms for cerebral vasodilation (in some areas) include hypoxemia, impaired autoregulatory response to increased blood pressure, and/or direct or indirect vascular effects of cocaine or its metabolites.
Animals↗
Effect of hypoxemia on the cardiovascular response to intracranial hypertension in postnatal lambs.
Large increases in intracranial pressure in fetal sheep result in more potent peripheral vasoconstriction and better maintenance of cerebral O2 consumption (CMRO2) than in postnatal sheep. The fetus is exposed to a lower PO2. We tested the hypothesis that low PO2 in postnatal lambs potentiates peripheral vasoconstriction and better maintains cerebral perfusion pressure and CMRO2. Pentobarbital-anesthetized lambs, 2-7 days old, were ventilated with either room air (n = 7) or a low O2 mixture to reduce arterial O2 saturation to 50% (n = 7). Elevation of intracranial pressure to within 3-5 mmHg of baseline mean arterial pressure for 30 min by ventricular fluid infusion initially caused a similar increase in arterial pressure in the normoxic [11 +/- 3 (SE) mmHg] and hypoxic (14 +/- 2 mmHg) groups. Plasma catecholamines increased more rapidly in the hypoxic group. However, plasma vasopressin levels were substantially elevated by hypoxia alone and failed to increase further with elevated intracranial pressure. Moreover, there was no significant difference between groups in the steady-state increase in arterial pressure, and microsphere-determined blood flow to intestines, kidney, skin, and muscle did not decrease in either group. Consequently, cerebral perfusion pressure, regional cerebral blood flow, and CMRO2 were reduced similarly in both groups. Therefore, hypoxemia failed to potentiate the postnatal pressor response. Low PO2 is unlikely to be the major mechanism for the potent Cushing response in the fetus.
Animals↗
Haemophilus influenzae type B impairment of pial vessel autoregulation in rats.
To examine the mechanisms of autoregulatory impairment in meningitis, we studied the effects of Haemophilus influenzae type b (Hib) on pial vessels during hemorrhagic hypotension in rats, using a cranial window technique. We prepared cranial windows in barbiturate-anesthetized, mechanically ventilated rats. Artificial cerebrospinal fluid or 10(5) Hib in cerebrospinal fluid (n = 7 each group) was suffused over the pial surface. Pial arteriolar diameter was measured hourly for 4 h. Autoregulation was assessed as the ability of pial arterioles to dilate in response to graded hemorrhagic hypotension at 2 and 4 h. Pial arterioles exposed to Hib dilated progressively to 149 +/- 27% of baseline at 4 h. Vessel diameter in the Hib group was significantly greater than in the control group beginning at 2 h. Autoregulation was progressively impaired in Hib-exposed rats compared with control rats [-5.85 +/- 1.38 versus -8.02 +/- 2.02 and -3.82 +/- 1.57 versus -8.53 +/- 1.72% dilation/kPa fall in mean arterial blood pressure at 2 and 4 h, respectively (p < 0.05)]. These data suggest that autoregulation is impaired in pial arterioles exposed to Hib because involved vessels have a finite dilatory capacity and are close to maximal dilation before hypotensive challenge.
Animals↗
Randomised controlled trial of day care for hypertension in pregnancy.
Our aim was to assess the effect of the introduction of a day-care unit on the care of women with non-proteinuric hypertension in pregnancy. A randomised controlled trial was carried out on 54 women who presented at 26 weeks of pregnancy or later with non-proteinuric hypertension (systolic blood pressure 150-170 mm Hg and/or diastolic pressure 90-105 mm Hg on two occasions at least 15 min apart). 30 women were allocated to care by the day unit and 24 were managed according to the established practice of their clinicians without access to the day unit (control group). Women in the control group spent on average 4.6 times longer as inpatients (difference in mean stay 4.0 days [95% confidence interval 2.1-5.9 days]) than the day-unit group and were 8.8 times (95% CI 3.0-25.8) more likely to be admitted to hospital. Induction of labour was 4.9 times (95% CI 1.6-13.8) more likely in the control than in the day-unit group and the development of proteinuria 11.4 times (95% CI 1.8-71.4) more likely. The control group had a mean of 1.5 fewer hospital outpatient visits (95% CI 0.36-2.64). The groups did not differ in their use of antihypertensive drugs. Day-unit care for hypertension in pregnancy significantly reduced the need for and the length of antenatal inpatient admissions and the number of medical interventions, at the cost of an increase in outpatient attendances. Our results are further evidence that inpatient care does not improve outcomes or prevent the development of proteinuria in this disorder.
Adult↗
Alternative splicing determines the carboxy terminus of the Epstein-Barr virus nuclear antigen 5 species expressed in the Burkitt's lymphoma cell line Daudi.
The Daudi strain of Epstein-Barr virus (EBV) possesses a genomic deletion, relative to the B95-8 EBV prototype, that removes the entire Epstein-Barr virus nuclear antigen 2 (EBNA2) open reading frame (ORF) and the sequences encoding the carboxy terminus of EBNA5. Immunoblot analysis carried out in this study indicates that two species of EBNA5 (31K and 37K) are expressed in Daudi cells. Nucleotide sequence analysis of Daudi cDNA clones has confirmed that, as a consequence of the genomic deletion, exons usually appearing further downstream in EBNA messages (exons U or HF) are spliced directly onto the truncated EBNA5 ORF. Furthermore, the use of alternative splicing suggests that the two EBNA5 species expressed in Daudi cells possess different carboxy termini.
Alternative Splicing↗
Circulatory dynamics during periodic intracranial hypertension in fetal sheep.
The human fetal head is periodically compressed during labor. The resulting increase in intracranial pressure (ICP) may exceed the hydrostatic increase in mean arterial pressure (MAP), thereby decreasing cerebral perfusion pressure (CPP). We determined whether the cardiovascular system of near-term fetal sheep is capable of rapidly increasing MAP during periodic increases in ICP. In 12 chronically instrumented fetuses, we produced sinusoidal oscillations in ICP with a maximum of 52 +/- 1 mmHg (baseline MAP) and a minimum of 4 +/- 1 mmHg at a 3-min periodicity by ventricular fluid infusion and withdrawal. Phasic increases in MAP and decreases in electromagnetically determined renal blood flow tracked behind ICP by 0.3-0.5 min. By the sixth cycle, tonic peripheral vasoconstriction that occurred attenuated by the reduction in CPP during subsequent ICP oscillations. By the 10th cycle, plasma catecholamines and vasopressin increased 20-fold. To more closely simulate the pattern during labor, we produced an ICP triangular pulse train with 5-min periodicity and pulse duration of 1.5 min in six other fetuses. The MAP response was nearly out of phase with this more rapid rise of ICP. Thus the phasic component of the fetal pressor response is inadequate for maintaining CPP when ICP is increased to baseline MAP in less than 0.75 min. However, when the ICP pulse duration and frequency are sufficiently high, a tonic pressor response that may be humorally mediated acts to minimize transient cerebral ischemia.
Animals↗
Oxygen free radicals and the cerebral arteriolar response to group B streptococci.
We used a cranial window preparation to observe the effects of direct application of group B streptococci to the surface of the brain in the adult rat. Continuous exposure to group B streptococci at concentrations of 10(3) and 10(5) organisms/mL caused progressive dilation of surface (pial) cerebral arterioles that became statistically significant (p less than 0.05) after 2.5 h. These results were reproduced with heat-killed organisms at the same concentration, but not with a bacteria-free filtrate of the growth medium. In separate studies, we found that infusion of alkaline cerebrospinal fluid (pH = 7.8) into the window did not reverse vasodilation, suggesting that it was not due to progressive cerebrospinal fluid acidosis. A solution of nitroblue tetrazolium infused into the window at the end of a 3-h exposure to the organism was promptly reduced, suggesting the presence of oxygen free radicals. Treatment with i.v. polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase in doses of 10,000 and 20,000 U/kg, respectively, was itself without effect on pial arterioles, but treatment with these compounds before exposure to group B streptococci eliminated the vasodilation. These data support a role for oxygen free radicals in the pathogenesis of pial arteriolar dysfunction induced by exposure to group B streptococci.
Animals↗
Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry.
A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.
Benzamides↗
Haemostatic measurements in childhood nephrotic syndrome.
Blood coagulation and platelet aggregation were assessed in children with nephrotic syndrome who were divided into the following groups: (1) relapse without treatment: (2) relapse on steroids; (3) early remission; (4) late remission and (5) steroid resistant. The renal histological findings were also recorded. Plasma anti-thrombin III (ATIII) levels were markedly reduced in groups 1 and 2, below normal in group 3 and were normal in groups 4 and 5. There was significant urinary loss of ATIII in groups 1 and 2 as well as in group 5. Plasma fibrinogen fluctuations exhibited the expected negative correlations with plasma ATIII. Reptilase time showed significant prolongation in groups 1, 2 and 3, and was near normal in groups 4 and 5. Platelet aggregation in response to arachidonic acid exhibited aggregation followed by disaggregation in groups 1, 2, 4 and 5, and was normal in group 3. Hyperaggregation in response to decreasing doses of ADP was noted in all patient groups as well as controls with no relationship to serum albumin levels. Aggregation responses to collagen and ristocetin were normal. It is concluded that: 1. The fluctuations in ATIII levels in childhood nephrotic syndrome are determined by the response to steroids and not by the renal histology per se. 2. An acquired fibrin polymerization defect (dysfibrinogenaemia) and an abnormality of the prostaglandin pathway of platelet activation, both reversible, are yet other haemostatic abnormalities in childhood nephrosis. 3. The discrepancies in the literature on haemostatic parameters, specially ATIII in childhood nephrosis, would not have arisen if their fluctuation in relation to steroid therapy as well as the renal histological features of nephrotic syndrome had been documented simultaneously.
Adolescent↗
New herpesviruses and their relevance for man.
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Journal Article↗
The genome of human herpesvirus 6: maps of unit-length and concatemeric genomes for nine restriction endonucleases.
More than 50 fragments resulting from complete digestion of the DNA of human herpesvirus 6 (HHV-6, strain U1102) with BamHI, EcoRI, HindIII, KpnI, NruI, SalI or SmaI have been isolated as clones in M13, plasmid, cosmid and lambda vectors. Using these clones, maps have been constructed for the fragments produced by nine restriction enzymes from unit-length virus genomes and from their concatemeric precursors. The unit-length genome is a linear, double-stranded molecule of 161.5 kbp composed of a central segment of a largely unique sequence of 141 kbp (U) with a sequence of 10 kbp duplicated in the same orientation at both 'left' and 'right' genomic termini (i.e. 'left' and 'right' copies of the direct repeat; DRL and DRR). Adopting as standard an orientation in which the major capsid protein gene is 'left' of the gene for alkaline exonuclease, then the 'right' genome termini and DRL. U junctions occur close to or within repetitive (GGGTTA)n sequences. Repetitions of short sequence motifs are present in at least two other regions of the genome. One of these regions consists of a simple repeat (TC/G) of approximately 1.5 kbp in length and is unstable as clones in bacterial vectors. The second region is stably maintained in such vectors and consists of a tandem array of at least 25 copies of a 110 bp sequence containing a single KpnI site. Comparisons of fragments arising from unit-length DNA with those from virus DNA from the nuclei of infected cells have shown that the concatemeric junctions in intracellular DNA contain head-to-tail dimers of the terminal duplications (i.e. ...U1.DRR1.DRL2.U2...). The gross structure established here for the genome from the U1102 isolate of HHV-6 resembles closely that suggested by Pellett and his colleagues for the Z29 isolate and differs from that of the five previously characterized human herpesviruses. This structure of HHV-6 DNA bears a superficial resemblance to that proposed for DNA from channel catfish virus and equine cytomegalovirus.
Base Sequence↗
Characterization of the DNA polymerase gene of human herpesvirus 6.
The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group.
Amino Acid Sequence↗
Efficacy of Cushing response during development in sheep.
Mean aortic pressure (MAP) increases (Cushing response) when intracranial pressure (ICP) approaches MAP. We elevated ICP to levels equivalent to normal baseline MAP with infusion of mock cerebrospinal fluid (CSF) into the lateral cerebral ventricles and contrasted responses in near-term fetal sheep, 1-wk-old lambs, and adult sheep anesthetized with pentobarbital sodium. With CSF infusion 1-wk-old lambs and adults produced sustained increases in MAP of 16 +/- 1 and 22 +/- 2 mmHg, respectively, over a 40-min period. However, cerebral blood flow fell 66 and 57%, and cerebral O2 uptake fell 34 and 37%, respectively. In the near-term fetus, MAP increased by 11 +/- 1 mmHg and cerebral blood fell 49% at 3 min of elevated ICP. However, by 15 min MAP had increased further (+17 +/- 2 mmHg) and cerebral blood flow was nearly restored. In contrast to postnatal sheep, cerebral O2 uptake was maintained throughout in the fetus. The mechanism of increased MAP differed among groups. In adults total peripheral resistance fell significantly, whereas in the fetus and lamb it remained constant. Cardiac output increased in each group, but, because of the fall in peripheral resistance, increased cardiac output was relatively more important to the rise in MAP in adults. In addition, marked vasoconstriction occurred in intestines and skin in the fetus. The Cushing response is well-developed in near-term fetal sheep. After birth it may lose its effectiveness in providing for the basal metabolic demand of the brain.
Aging↗
Cerebral blood flow response to hypercapnia in immature fetal sheep.
We have reported recently that the cerebral blood flow (CBF) response to isocapnic hypoxic hypoxia is blunted in fetal sheep in utero at 93 days of gestation (term = 145-150 days), a time of rapid brain differentiation in this species. Cerebral O2 transport fell rather than being maintained, as it is in more mature fetuses. The reason for the blunted response was not clear. We hypothesized that the CBF response to hypercapnia also might be blunted. We studied 10 immature fetal sheep in utero at a mean gestational age of 92 days 24 h after catheters were placed into the superior sagittal sinus, axillary artery, and inferior vena cava. We raised the fetal arterial carbon dioxide tension (PaCO2) by changing the mother's inspired PCO2. CBF was measured before and during hypercapnia by the microsphere method. The overall increase in CBF in response to hypercapnia in immature fetuses was lower than in near-term fetuses. However, the difference was eliminated after correcting for differences in cerebral O2 consumption. This study failed to show any defect in the ability of cerebral vessels in immature fetal sheep to respond to carbon dioxide.
Animals↗
Pulmonary disease in systemic urticarial vasculitis: the role of bronchoalveolar lavage.
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Adult↗