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D Robertson

Publications and source records attributed to D Robertson.

At least 181 records · Page 10Linked to original sources
PubMed1996

Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.

UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40,000. Km values were measured for UDP-glucose as 5.5 +/- 1.4 mM and for NAD+ as 20 +/- 3 microM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

Alcohol Dehydrogenase↗
PubMed1996

cis-Diamminedichloroplatinum(II)-induced cell death through apoptosis in sensitive and resistant human ovarian carcinoma cell lines.

We have studied the effects of the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-platin) on three human ovarian carcinoma cell lines - one sensitive to the drug (CH1), one with acquired resistance (CH1cisR) and one with intrinsic resistance (SKOV-3). Previous work has shown that the 50% inhibitory concentrations (IC50 values) after a 2-h exposure to the drug are: CH1, 2.5 microM; CH1cisR, 7.5 microM; and SKOV-3, 33 microM. Despite the variation in sensitivity, the amount of Pt bound to DNA and the rate of removal of Pt was similar for the three lines. There were significant differences in the rates of formation of DNA cross-links but these were not large enough to account for the high resistance of the SKOV-3 line. We have reported that in the L1210 murine leukaemia cell line there are two mechanisms of cisplatin-induced cell death - one of which involves apoptosis. In this paper, we report on an investigation into whether sensitivity to apoptosis played a role in the resistance of these ovarian lines toward cisplatin. After a 2-h incubation with the drug, cells from the three lines showed evidence of death through apoptosis. The cells detached from the culture dish in a time- and dose-dependent fashion. These cells morphologically were quite distinctive from the attached cells and showed changes in their chromatin structure indicative of apoptosis. Their DNA had not been degraded into oligonucleosomal fragments (200 bp and multiples thereof) but had been cut into larger fragments (30 kilobase pairs, kbp) of a size associated with chromatin domains (chromatin loops). At equitoxic doses of drug, the quantity of cells undergoing apoptosis was similar for the three cell lines. The most prominent effect on cell-cycle kinetics was a slowdown in S-phase transit during which the cells underwent apoptosis. Cells that successfully completed the S phase subsequently suffered a temporary G2 block. We propose that the sensitivity of these cell lines to cisplatin was governed by their ability to handle damage caused by platination of the DNA and that the major mechanism of cisplatin-induced cell death in all three cell lines was the induction of apoptosis.

Antineoplastic Agents↗
PubMed1996

Apoptotic and non-apoptotic cell death induced by cis and trans analogues of a novel ammine(cyclohexylamine)dihydroxodichloroplatinum(IV) complex.

It has been previously demonstrated that cisplatin induces apoptosis in the CH1 human ovarian carcinoma cell line. This study demonstrates that two novel platinum (Pt) analogues JM149 and JM335, which are the cis and trans geometry respectively of ammine(cyclohexylamine)dihydroxodichloroPt(IV), initiate apoptosis in this cell line at physiologically relevant concentrations (IC50 values 2 h drug exposure were 35.3 microM for JM149 and 18.7 microM for JM335). While at equimolar drug concentrations there was a 2-fold higher level of total platinum-DNA adducts following exposure to JM335 vs JM149, at equitoxic concentrations, levels were similar (80 vs 70 pmol Pt mg-1 DNA respectively). Following a 2 h incubation with 2 x IC50 of both drugs, cells rounded up and detached in a time-dependent manner but with the kinetics of apoptosis being more rapid for JM335. The majority of detached cells exhibited morphology associated with apoptosis which was further supported by the presence of a 50 kb fragment detected in DNA lysates prepared from these cells. JM149 induced apoptosis across a range of concentrations (2 x, 5 x and 10 x IC50) with a 50 kb DNA fragment being detected at all concentrations. However, in marked contrast to this, JM335 failed to cause apoptosis at 10 x IC50, the detached cells neither displaying apoptotic morphology nor a detectable 50 kb DNA fragment. Moreover, these detached cells showed evidence of extensive vesiculation while the DNA remained normal in appearance and thus appeared to have died by a non-apoptotic mode. Apoptosis also appeared to be induced to a lesser extent at 5 x IC50 of JM335 as demonstrated by a less intense 50 kb fragment compared with that seen at 2 x IC50. The main cell cycle effect of these drugs (at 2 x IC50) was a slowdown in S-phase traverse during which most but not all of the apoptosis appeared to occur. However, at 5 x IC50 of JM335 cells appeared frozen in all phases of the cell cycle with little progress from G1 to S accompanied by a build-up of cells in G2 indicative of a G2/M block. This difference in cell cycle effect may account for the reduced level of apoptosis at this concentration and a failure to engage apoptosis at higher concentrations. These data suggest that the nature of the platinum drug (and consequently, the nature of resultant DNA damage) may have important implications in determining the rate and mechanism of cell death in this cell line. The cell death effects observed with the trans complex JM335 may correlate with the induction of DNA single-strand breaks in this cell line.

Antineoplastic Agents↗
PubMed1996

Clinical models of cardiovascular regulation after weightlessness.

After several days in microgravity, return to earth is attended by alterations in cardiovascular function. The mechanisms underlying these effects are inadequately understood. Three clinical disorders of autonomic function represent possible models of this abnormal cardiovascular function after spaceflight. They are pure autonomic failure, baroreflex failure, and orthostatic intolerance. In pure autonomic failure, virtually complete loss of sympathetic and parasympathetic function occurs along with profound and immediate orthostatic hypotension. In baroreflex failure, various degrees of debuffering of blood pressure occur. In acute and complete baroreflex failure, there is usually severe hypertension and tachycardia, while with less complete and more chronic baroreflex impairment, orthostatic abnormalities may be more apparent. In orthostatic intolerance, blood pressure fall is minor, but orthostatic symptoms are prominent and tachycardia frequently occurs. Only careful autonomic studies of human subjects in the microgravity environment will permit us to determine which of these models most closely reflects the pathophysiology brought on by a period of time in the microgravity environment.

Autonomic Nervous System↗
PubMed1996

The effect of inhaled nitric oxide in pediatric asthma.

Nitric oxide (NO) appears to play an important role in regulating several biologic functions in the lung, including modulation of pulmonary arterial and bronchial smooth muscle tone. Recent studies have shown that relatively high concentrations of inhaled NO reduce the bronchoconstrictor effect of methacholine in animal models. This raises the possibility that NO inhalation might have therapeutic potential as an alternative bronchodilator. Although investigation of this potential in adults with airway reactivity or bronchial asthma has been reported, data are lacking on the role of NO in the pediatric asthma population. We therefore performed spirometry on 12 children with asthma (mean age 11.1 yrs) at baseline (B), immediately after inhaling 40 ppm NO (NO-1), 10 min after inhaling NO (NO-10), and after inhalation of a standard beta 2-agonist, albuterol (A). Baseline pulmonary functions (% predicted +/- SEM) were FVC of 103.2 +/- 5.6, FEV1 of 82.2 +/- 3.3, FEF-max of 97.0 +/- 3.6, and FEF25-75% of 53.5 +/- 3.3. There were no statistically significant differences between baseline and NO-1 or NO-10 between any of the four pulmonary function parameters measured. Inhaled albuterol, however, resulted in significant improvement (% predicted +/- SEM) in FVC to 109.8 +/- 3.5, FEV1 to 99.7 +/- 2.9, FEFmax to 106.5 +/- 5.1, and FEF25-75% to 84.4 +/- 6.4 compared with the baseline and NO inhalation groups. We conclude that NO inhaled at 40 ppm has no apparent bronchodilatory effect in pediatric subjects with asthma and mild airways disease. The clinical application of this gas as a therapeutic modality under these conditions is questionable.

Administration, Inhalation↗
PubMed1996

Biological and immunological characterization of inhibin forms in human plasma.

A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B.

Animals↗
PubMed1995

Cell wall polysaccharide biosynthesis and related metabolism in elicitor-stressed cells of French bean (Phaseolus vulgaris L.).

Enzyme activities involved in quantitative and qualitative flux of sugars into cell wall polysaccharides were determined following elicitor treatment of suspension cultured cells of French bean (Phaseolus vulgaris L.). Two subsets of activities were examined: the first were involved in synthesis and metabolism of UDP-glucose and the provision of the pool of UDP-sugars, and the second a selection of membrane-bound glycosyltransferases involved in the synthesis of pectins, hemicelluloses and glucans of the primary cell wall. Of the first group, only UDP-glucose dehydrogenase (EC 1.1.1.22) showed any significant induction in response to elicitor treatment, sucrose synthase (EC 2.4.1.13), UDP-glucuronate decarboxylase (EC 4.1.1.35), UDP-glucose and UDP-xylose 4-epimerases (EC 5.1.3.2 and EC 5.1.3.5 respectively) did not change in activity significantly over the time course. In contrast, enzymes of the second group showed a more complex response. Callose synthase (glucan synthase II, EC 2.4.1.12) increased in activity, as has been shown in other systems, while arabinan synthase (EC 2.4.1.-), xylan synthase (EC 2.4.1.72), xyloglucan synthase (EC 2.4.1.72) and glucan synthase I (EC 2.4.1.12) activities were rapidly depleted from membranes within 3 h following elicitor action. This rapid turnover of activity was striking, indicating that the half-life of such enzymes can be short and that elicitor action causes substantial perturbation of some membrane activities. Glucan synthase I activity appears to increase in the later stages over the time period measured, indicating some recovery of this metabolism.

Cell Membrane↗
PubMed1995

Dystonia and unique muscle features. A 23-year follow-up and correction of diagnosis in two brothers.

OBJECTIVE: To provide follow-up information and a corrected diagnosis on two brothers who were primarily described in the ARCHIVES in 1971 as having had a genetic dystonia with unusual muscle biopsy features. MEASURES: Clinical observation of response to treatment and muscle histologic findings. RESULTS: These brothers are an unusual example of dopa-responsive dystonia that was present since birth. The muscle histopathologic features were caused by an abnormal cerebral influence on the developing motor unit and were not a primary abnormality. A repeated muscle biopsy performed 1 year after treatment continued to show the same pattern of fiber-type abnormalities. CONCLUSIONS: Dopa-responsive dystonia can be present from birth or early infancy. The response to levodopa is excellent even after a delay in treatment of more than 20 years. Intrauterine dystonia can cause a predominance of small type 2 fibers. A trial of levodopa/carbidopa is indicated in all patients with a childhood-onset dystonia or gait disturbance.

Adult↗
PubMed1995

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.).

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H2O2. The cells recover and start producing ATP immediately of H2O2 production. This indicates that the increased O2 uptake is primarily incorporated into active O2 species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M(r) 40,000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.

Adenosine Triphosphate↗
PubMed1995

Effect of neurovestibular stimulation on autonomic regulation.

Conditions associated with nausea and vomiting, such as motion sickness or side effects of medications, are commonly associated with a clinical picture consistent with parasympathetic activation and sympathetic withdrawal. It can be postulated, therefore, that vestibular stimulation contributes to sympathetic withdrawal. To test this hypothesis five normal volunteers, 24-33 years old, were studied during caloric vestibular stimulation while monitoring muscle sympathetic nerve activity directly through a needle electrode placed in a peroneal nerve. The ear was irrigated with water at a flow rate of 450 ml/min and 37 degrees C. The water temperature was sequentially lowered by 7 degree C intervals until intolerable side effects developed or a temperature of 16 degrees C was reached. Nystagmus was induced in all subjects, but heart rate, blood pressure, muscle sympathetic nerve activity and plasma norepinephrine levels did not change significantly during or after caloric stimulation, even when the subjects felt dizzy and nauseated. No evidence of sympathetic withdrawal was observed in any subject either by muscle sympathetic nerve activity or plasma norepinephrine measurements. In conclusion, we have found that selective vestibular stimulation is not accompanied by significant changes in the sympathetic nervous system function. In particular, no sympathetic withdrawal was observed. It could be argued that lack of sympathetic stimulation is an inadequate response to the symptoms associated with caloric stimulation.

Adult↗
PubMed1995

The behaviour of the f2-f1 acoustic distortion product: lack of effect of brainstem lesions in anaesthetized guinea pigs.

Two tones of frequency f1 and f2 (the primary tones), when presented simultaneously to the ear, generate acoustic distortion products in the external ear canal. One of these distortion products, of frequency f2-f1, has been shown to undergo a reversible change in amplitude when the primary tones generating distortion are presented continuously to the test (ipsilateral) ear (Brown, 1988; Kirk and Johnstone, 1993). The effect is apparent for low and moderate primary tone intensities and has been postulated to be caused by the action of a neural feedback loop via the superior olivary complex. We have carried out a series of studies of this phenomenon in anaesthetized guinea pigs, making brainstem lesions positioned so as to interrupt the known medical and/or lateral efferent projections to the cochlea from the superior olivary complex. We could not demonstrate any consistent change after lesioning, either in the baseline level of f2-f1 or in the alteration of f2-f1 caused by continuous monaural primary tones. These results are not consistent with the suggestion by others that a neural feedback loop involving either the medial (Brown, 1988) or lateral (Kirk and Johnstone, 1993) olivocochlear efferents may be responsible for the effect. We therefore conclude that either 1) the changes in f2-f1 produced by continuous low-level primary tones reflect the operation of intrinsic hair cell mechanisms and do not involve efferent feedback via brainstem nuclei or 2) a neural feedback loop does play a role, but this loop involves an unknown pathway that was not interrupted by our lesions.

Acoustic Stimulation↗
PubMed1995

Neurotransmitter and neuromodulator systems of the rat inferior colliculus and auditory brainstem studied by in situ hybridization.

This study was concerned with the distribution of a variety of putative neuromodulator and neurotransmitter systems in auditory regions of the rat brainstem using in situ hybridization histochemistry. Serial brain sections were screened for the presence of mRNAs for (i) precursors of the neuroactive substances cholecystokinin, somatostatin, proenkephalin and substance P (preprotachykinin), (ii) glutamic acid decarboxylase, the key synthesizing enzyme for GABA, or (iii) subunits alpha 1, alpha 2 and alpha 3 of the GABAA receptor. Detectable message for all of these probes was found in at least one auditory brainstem area. There were clear differences in the distribution of the various mRNAs in subregions of the inferior colliculus, superior olivary complex, lateral lemniscus and cochlear nucleus. Cells expressing mRNA for glutamic acid decarboxylase were most prominent in the inferior colliculus, but were also present in all lower auditory brainstem nuclei, except the medial superior olivary nucleus and medial nucleus of trapezoid body. The mRNA for GABAA alpha 1 receptor subunits was detectable in all auditory regions investigated, although at different levels of expression. GABAA alpha 2 and alpha 3 mRNA signals were seen in inferior colliculus, lateral lemniscus and in almost all superior olivary complex regions, but in fewer cells and at lower levels than the GABAA alpha 1 subtype. Moderate to high levels of preprocholecystokinin mRNA expression were seen in all subregions of the inferior colliculus. In other auditory brainstem areas, preprocholecystokinin mRNA levels were either low or absent. With regard to mRNAs for the neuroactive peptides somatostatin, preprotachykinin and preproenkephalin, all were expressed in the inferior colliculus but there were differences in their cellular distribution. For example, there were almost no preprotachykinin mRNA expressing cells in the central nucleus of inferior colliculus and levels of somatostatin mRNA were especially high in the dorsal cortex and in layer 3 of the external cortex of inferior colliculus. There were also differences in the pattern of expression of these mRNAs in the various brainstem auditory nuclei; there was no preprotachykinin mRNA in any part of the superior olivary complex, only somatostatin mRNA was found in the ventral cochlear nucleus, and expression of preproenkephalin mRNA was pronounced in the ventral nucleus of the trapezoid body and the rostral periolivary zone. The data are considered in light of the connectivity and functional organization of the auditory brainstem.

Animals↗
PubMed1995

Programmed cell death in response to chemotherapeutic agents in human germ cell tumour lines.

Testicular germ cell tumours are amongst the most chemosensitive neoplasms both in vivo and in vitro. In the present study we demonstrate that following exposure to drugs used in chemotherapeutic treatment of testicular germ cell cancer tumour cells undergo death by apoptosis. Thus, after exposure of the GCT27 embryonal carcinoma cell line to cisplatin, we observed the degradation of DNA into oligonucleosomal fragments, which is a hallmark of apoptosis. Furthermore, light, fluorescence and electron microscopy reveal the presence of condensed abnormal shaped nuclear chromatin which is characteristic of apoptosis. Changes diagnostic of apoptosis were also observed following (a) cisplatin treatment of the GCT48 and Susa embryonal carcinoma cell lines and the GCT44 yolk sac tumour cell line and (b) etoposide treatment of the GCT27 and Susa cell lines. When the GCT27 cell line was treated with 15 microns cisplatin, apoptosis was first observed at 6-9 h and greater than 90% of cells were dead within 24 h. Apoptosis was not blocked when cisplatin-treated cells were incubated in the presence of cycloheximide, although this agent did cause a 4-6 h delay in the onset of cell death. In addition, we demonstrated that the GCT27 cell line can be induced to undergo apoptosis by exposure to low concentrations of the calcium ionophore, ionomycin. These observations show that germ cell tumours are remarkably sensitive to a range of agents that act by different mechanisms. They are triggered to undergo apoptosis rapidly by a mechanism that is not blocked by inhibitors of protein synthesis.

Apoptosis↗
PubMed1995

Urinary vascular endothelial growth factor concentrations in women undergoing gonadotrophin treatment.

A recently identified cytokine, vascular endothelial growth factor (VEGF, vascular permeability factor) has been implicated in ovarian hyperstimulation syndrome in women undergoing assisted reproduction. We postulate that circulating and urinary VEGF values increase following gonadotrophin stimulation, in parallel with the increased ovarian vascularity. A VEGF radioimmunoassay was developed using iodinated VEGF as tracer, a goat anti-VEGF serum as antiserum and recombinant human VEGF as standard. The specificity of the assay was confirmed by comparing the reverse phase high-performance liquid chromatography (HPLC) pattern of VEGF immunoactivity in urine and urine spiked with recombinant VEGF. Urine was concentrated 5-fold prior to measurement by the radioimmunoassay. VEGF:creatinine ratios in early morning urine samples were used to monitor daily urinary VEGF concentrations based on its high correlation (r = 0.77, P < 0.001) with VEGF concentrations in 24 h urine collections. No diurnal variation in VEGF:creatinine ratios was detected. VEGF:creatinine ratios were determined daily from nine women undergoing gonadotrophin-releasing hormone (GnRH) agonist/gonadotrophin treatment. In a further 16 women, early morning urine samples were collected in the peri-ovulatory period. A significant increase (P < 0.005, n = 25) was observed in VEGF:creatinine ratios following human chorionic gonadotrophin (HCG) administration. VEGF:creatinine ratios correlated poorly (r < 0.34) with plasma oestradiol, follicle number and size. It is concluded that urinary VEGF/creatinine ratios increase following HCG stimulation.

Chorionic Gonadotropin↗
PubMed1995

Orthostatic hypotension: epidemiology, pathophysiology and management.

Orthostatic hypotension is characterized by low upright blood pressure levels and symptoms of cerebral hypoperfusion. Whereas orthostatic hypotension is heterogeneous, correct pathophysiologic diagnosis is important because of therapeutic and prognostic considerations. Although therapy is not usually curative, it can be extraordinarily beneficial if it is individually tailored. Management of the Shy-Drager syndrome (multiple-system atrophy) remains a formidable challenge.

Humans↗