Biomedical subjects

R P Ekins

Publications and source records attributed to R P Ekins.

At least 19 recordsLinked to original sources

PubMed2001

Thyroid hormone receptor expression in rat placenta.

The expression of c- erbAalpha and -beta encoded thyroid hormone receptors (TR) was investigated in rat placenta between 16 and 21 days of gestation (dg), and in fetal liver and brain at 16 dg, using semi-quantitative RT-PCR and nuclear 3,5,3'-triiodothyronine (T(3)) binding. TRalpha1, TRbeta1, c- erbAalpha 2 and c- erbAalpha 3 mRNA abundance was unchanged in placenta between 16 and 21 dg, as was the dissociation constant (K(d)) of T(3) binding. The maximal T(3) binding capacity (B(max)) in placenta doubled over this period, suggesting placental TR binding activity is post-transcriptionally regulated. Transcript abundance in tissues at 16 dg can be summarized: TRalpha1, placenta=fetal liver fetal brain; c- erbAalpha 2 and alpha3, placenta=fetal liver<fetal brain; TRbeta2; none detected. T(3)binding in fetal liver and brain exhibited equivalent K(d) and B(max), the K(d) being less than 50 per cent of that in placenta, though B(max) was unchanged. The higher K(d)in placenta may reflect tissue-specific patterns of TR modification. In conclusion, rat placenta expresses significant levels of c- erbAalpha and -beta transcripts and protein, providing a possible mechanism of action for T(3) of maternal and fetal origin in this tissue.

Animals↗

PubMed2000

Maternal thyroid status regulates the expression of neuronal and astrocytic cytoskeletal proteins in the fetal brain.

Maternal thyroid hormone (TH) crosses the placenta and is postulated to regulate fetal brain development. However, TH-dependent stages of fetal brain development remain to be characterised. We have therefore compared the levels of several neuronal and glial cytoskeletal proteins in fetal brains from normal (N) and partially thyroidectomised (TX) rat dams by immunoblotting. Pregnancies were studied both before and after the onset of fetal TH secretion, which occurs at 17.5 days gestation (dg) in the rat. Maternal hypothyroidism disrupted fetal growth, so that fetal body and brain weights were reduced near term. Vimentin expression was unaffected, however, indicating normal acquisition of neuronal and glial precursor cells. Fetal brain levels of glial fibrillary acidic protein (GFAP) were reduced at 21 dg, suggesting delayed astrocytic differentiation, although regression analysis demonstrated appropriate GFAP levels for brain weight. Levels of alpha-internexin, the earliest neurofilament protein expressed in fetal brain were reduced at 16 dg in TX dams, but increased at 21 dg. The ontogeny of neurofilament-L was also perturbed in these pregnancies, with deficient levels apparent at both 16 and 21 dg. These effects on neuronal cytoskeletal proteins were unrelated to fetal brain growth retardation. These findings confirm that maternal hypothyroidism disrupts early fetal brain development. Early disturbances in neuronal differentiation are not corrected by the onset of fetal TH secretion. Such disturbances may contribute to the neurological damage observed in children born to hypothyroxinaemic mothers.

Analysis of Variance↗

PubMed1999

SHORT COMMUNICATION maternal thyroid dysfunction and c- fos and c- jun expression in rat placenta.

Maternal thyroid dysfunction is associated with perturbed fetal brain development and neurological deficits in adulthood in rat and human. To investigate whether these effects occur secondary to placental dysfunction, c- fos and c- jun expression in placenta from normal (euthyroid) and moderately hypothyroid rat dams were investigated by Northern hybridization analysis. In normal placenta, c- fos expression increased by 74 per cent between 16 and 21 days of gestation (dg) whereas c- jun expression declined by 46 per cent. Moderate maternal hypothyroidism depressed placental c- fos expression by 32 per cent at 19 dg, but elevated c- fos and c- jun expression by 139 and 86 per cent, respectively, at 21 dg. Maternal hypothyroidism may therefore induce c- fos/c- jun -related placental dysfunction, but only relatively late in gestation when fetal thyroid function is already established.

Animals↗

PubMed1999

Maternal hypothyroxinemia disrupts neurotransmitter metabolic enzymes in developing brain.

Maternal thyroid status influences early brain development and, consequently, cognitive and motor function in humans and rats. The biochemical targets of maternal thyroid hormone (TH) action in fetal brain remain poorly defined. A partially thyroidectomized rat dam model was therefore used to investigate the influence of maternal hypothyroxinemia on the specific activities of cholinergic and monoaminergic neurotransmitter metabolic enzymes in the developing brain. Maternal hypothyroxinemia was associated with reduced monoamine oxidase (MAO) activity in fetal whole brain at 16 and 19 days gestation (dg). A similar trend was observed for choline acetyltransferase (ChAT) activity. In contrast, DOPA decarboxylase (DDC) activity was markedly elevated at 21 dg. Further study of these enzymes at 14 dg showed no differences between normal and experimental progeny - suggesting they become TH sensitive after this age. Tyrosine hydroxylase (TyrH) and acetylcholinesterase (AChE) activities were unaffected prenatally. During postnatal development, the activities of TyrH, MAO, DDC and, to a lesser extent, AChE were increased in a brain region- and age-specific manner in experimental progeny. The prenatal disturbances noted in this study may have wide-ranging consequences since they occur when neurotransmitters have putative neurotropic roles in brain development. Furthermore, the chronic disturbances in enzyme activity observed during postnatal life may affect neurotransmission, thereby contributing to the behavioural dysfunction seen in adult progeny of hypothyroxinemic dams.

Aging↗

PubMed1999

Maternal hypothyroxinemia influences glucose transporter expression in fetal brain and placenta.

The influence of maternal hypothyroxinemia on the expression of the glucose transporters, GLUT1 and GLUT3, in rat fetal brain and placenta was investigated. Fetal growth was retarded in hypothyroxinemic pregnancies, but only before the onset of fetal thyroid hormone synthesis. Placental weights were normal, but placental total protein concentration was reduced at 19 days gestation (dg). Immunoblotting revealed a decreased abundance of GLUT1 in placental microsomes at 16 dg, whereas GLUT3 was increased. Fetal serum glucose levels were reduced at 16 dg. In fetal brain, the concentration of microsomal protein was deficient at 16 dg and the abundance of parenchymal forms of GLUT1 was further depressed, whereas GLUT3 was unaffected. Northern hybridization analysis demonstrated normal GLUT1 mRNA levels in placenta and fetal brain. In conclusion, maternal hypothyroxinemia results in fetal growth retardation and impaired brain development before the onset of fetal thyroid function. Glucose uptake in fetal brain parenchyma may be compromised directly, due to deficient GLUT1 expression in this tissue, and indirectly, as a result of reduced placental GLUT1 expression. Though corrected by the onset of fetal thyroid hormone synthesis, these deficits are present during the critical period of neuroblast proliferation and may contribute to long term changes in brain development and function seen in this model and in the progeny of hypothyroxinemic women.

Animals↗

PubMed1998

Ligand assays: from electrophoresis to miniaturized microarrays.

The main developments in the "ligand assay" field in which I have been involved are traced. These include the original development of "first generation" competitive assays relying on radiolabeled analyte markers; the development of the first "second generation", noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first "third generation" miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter--applicable both to immunoassay and to DNA/RNA analysis--are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.

Electrophoresis↗

PubMed1994

Developing multianalyte assays.

Multianalyte 'binding' assays represent a major advance in microanalytical technology for the measurement of substances of biological importance. Their further development should facilitate sensitive and reproducible quantification of analytes in many areas that are currently problematic, including diagnostic medicine and the standardization of biologicals.

Forecasting↗

PubMed1994

Transport of thyroid hormones to target tissues.

Endemic iodine deficiency is associated with maternal hypothyroxinemia and a relatively high incidence of neurological disorders in the offspring. The previous assumption that the placenta is impermeable to maternal thyroid hormone, has resulted in the erroneous suggestion that iodine per se has an essential role in brain development. Furthermore, the observed factorial rise in thyroxine-binding globulin (TBG) in pregnancy has often been misinterpreted as preventing thyroid hormone loss to either the fetal compartment or excretory systems. However, physiochemical analysis of the role of specific binding proteins in hormone delivery, combined with epidemiological evidence and evolutionary considerations has led us to postulate that a) maternal thyroxine (T4) is transported to the fetus, and is of crucial importance in early fetal development, and b) TBG forms part of a control system specifically designed to maintain at an optimal level the T4 environment to which the developing fetus is exposed. Placental transfer of maternal T4 in a variety of mammalian species (including humans) is now well established. Further experimental studies in rats have shown that perturbation of the intrauterine thyroid hormone environment during critical phases of brain development results in a spectrum of biochemical dysgenesis. For example, in fetal brains deriving from hypothyroxinemic (Tx) rat dams, severe disruption of phosphate metabolism is observed and the ontogenesis of two enzyme activities associated with growth control, protein kinase C and ornithine decarboxylase, are compromised. Development of brain function is also impaired, as evidenced by the dysgenesis of certain neurotransmitter metabolic activities (choline acetyltransferase and DOPA decarboxylase).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

New perspectives in radioimmunoassay.

In addition to providing the basis of 'ultrasensitive' immunoassays, nonisotopic labels permit development of 'user friendly' methodologies, including simple devices designed for extralaboratory use and intended to provide yes/no answers to basic diagnostic questions, for example, the presence of an infectious disease, the commencement of pregnancy etc. Immunosensors are also intended to fulfil these objectives (albeit yielding quantitative information), but despite much research the prospect of adequately sensitive instruments of this type remains remote. Finally, nonisotopic labels enable the development of a new generation of miniaturized multianalyte assays permitting in principle the determination of entire analyte menus in small samples of body fluids. Such technologies will resemble those employed in compact disk recorders and other data-storage devices, differing only in that light emitted from discrete zones on a solid surface is fluorescent (or possibly chemiluminescent) rather than reflected, and yields chemical rather than physical information. The emergence of such technologies, will, inter alia, facilitate the screening of entire populations for infectious disease, endocrine dysfunctions, genetic abnormalities etc. using, for example, finger-tip blood samples, throat and endocervical swabs, and hopefully will contribute to the large-scale diagnosis and ultimate reduction of disease.

Humans↗

PubMed1993

Hypothyroidism in the adult rat causes brain region-specific biochemical dysfunction.

The influence of hypothyroidism in the adult rat on brain biochemistry was investigated. Hypothyroidism was induced in 6-month-old male rats by partial thyroidectomy coupled with the administration of 6-n-propyl-2-thiouracil (0.005%, w/v) in the drinking water. Age-matched euthyroid males served as the controls. Hypothyroidism resulted in brain region-specific changes in certain catabolic enzyme activities. Acid phosphatase activity was reduced in the cerebellum (by 34%) and the medulla (by 38%), whereas alkaline phosphatase activity was decreased in the midbrain (by 37%) and the subcortex (by 49%). A differential response was also observed in the case of aryl sulphatase activity: aryl sulphatase A (myelin-degradative activity) was diminished in the cerebellum (by 56%), whereas aryl sulphatase B remained unchanged in all regions. Acetylcholine esterase activity was reduced in the cerebellum (by 45%), the medulla (by 34%) and the subcortex (by 45%), whereas monoamine oxidase activity was affected in only one region, the cerebellum, where it was increased by (61%). The compromise of myelin and neurotransmitter degradative enzyme activities may place severe restrictions on normal brain function. The vulnerability of the adult rat cerebellum to the effects of thyroidectomy is commensurate with the known clinical signs of cerebellar dysfunction in adult hypothyroid man. These findings raise the possibility of an important role for the thyroid hormones in the mature brain.

Acetylcholinesterase↗

PubMed1993

The influence of the maternal thyroid hormone environment during pregnancy on the ontogenesis of brain and placental ornithine decarboxylase activity in the rat.

The influence of maternal hypothyroxinaemia on early brain and placental development was examined in a partially thyroidectomized (parathyroid-spared; TX) rat dam model. Ornithine decarboxylase (ODC) specific activity, along with more general indices of cell growth, were determined in prenatal whole brain (at 15, 19 and 22 days of gestation), postnatal brain regions (at 5, 10 and 14 days) and placenta. Maternal hypothyroxinaemia resulted in reductions in fetal body weight, brain weight, brain DNA content and brain total protein content at 15 days of gestation; the latter effect persisting until 19 days of gestation. Further changes in brain cell growth were observed near term, when an increase in the DNA concentration was accompanied by a decrease in the total protein:DNA ratio. Growth of the postnatal brain regions appeared normal, with the exception of an isolated increase in the protein content of the cerebellum at postnatal day 5. Determination of the specific activity of brain ODC revealed a complex pattern of change in the progeny of TX dams, superimposed upon the normal ontogenetic decline. In the fetal brain, activity was initially deficient at 15 days of gestation but was increased at 22 days of gestation relative to controls. The compromise extended into the postnatal period; ODC specific activity being transiently reduced in the brainstem, the subcortex and the cerebral cortex. Placental development was less consistently affected; wet weight, gross indices of cell growth (DNA content, DNA concentration, total protein:DNA ratio) and ODC specific activity were all normal in the TX dam. However, cytosolic and total protein concentrations were reduced at 15 and 19 days of gestation respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Maternal hypothyroxinemia and brain development: I. A hypothetical control system governing fetal exposure to maternal thyroid hormones.

Maternal hypothyroxinemia consequent on endemic iodine deficiency is associated with an increased incidence of neurological disorders in the offspring. Such correlations were originally postulated as reflecting direct effects of elemental iodine on fetal brain development during early pregnancy, it being generally believed that maternal thyroid hormones do not cross the placenta in significant amount in consequence of the presence of elevated concentrations of TBG in maternal blood. However TBG possesses the capacity to enhance T4 transport to particular target organs during pregnancy. This realization led us to hypothesize a) that maternal T4 is transported to the fetus, and is of crucial importance to early fetal development, and b) that TBG forms part of a control system specifically designed to maintain at an optimal level the T4 environment to which the developing fetus is exposed. Subsequent studies in rats demonstrated that maternal T4 traverses the placenta in significant amounts prior to the development of the fetal thyroid. Other studies have led us to suggest that one or more isoforms of HCG may be implicated in a feed-back system interacting with the hypothalamic/pituitary system governing maternal thyroid hormone secretion. Though our experimental work has primarily focused on the effects of thyroid hormones on the fetal brain, we believe it to be likely that fetal exposure to maternal hormones is under placental control, and that other components of this putative system are worthy of study.

Animals↗

PubMed1992

Maternal hypothyroxinemia and brain development: II. Biochemical, metabolic and behavioural correlates.

Using a rat model, we have investigated the influence of maternal hypothyroxinemia throughout pregnancy on brain development in young and adult progeny. Although no consistent change was observed in whole brain total protein concentration, the subcellular distribution of protein was adversely affected. Isolation of glycoprotein from developing brain by concanavalin A-affinity chromatography and subsequent resolution by gel electrophoresis revealed the selective compromise of particular glycoprotein species. Furthermore, both control and experimental progeny expressed unique glycoprotein species which either persisted over the period studied or were transient. Calcineurin, a regulator of neurite elongation, was compromised in young progeny, as were a number of lysosomal enzymes (beta-D-glucosidase and aryl sulphatase). In adult progeny, the content of cerebroside sulphate (a major myelin galactolipid) was reduced in midbrain and paleocortex, and brain region-specific compromise was observed for acetylcholine metabolic enzymes. These changes were associated with alterations in behavioural output. We conclude that the availability of maternal thyroxine to the fetus may be a critical determinant for normal brain development and function.

Animals↗

PubMed1991

Pregnancy-induced changes in thyroid function: role of human chorionic gonadotropin as putative regulator of maternal thyroid.

Marked changes in maternal thyroid activity occur in pregnancy. It has been suggested that hCG may stimulate maternal T4 secretion, given its in vitro thyrotropic activity ascribed to a significant degree of structural homology with TSH. In a longitudinal study of 32 normal pregnant women, we attempted to clarify the functional activity of the thyroid in early and late pregnancy and the possibility of a nonpituitary control on the thyroid. Total T4 and T4-binding globulin levels were increased from the first trimester onward. Free T4 levels did not differ in the first trimester from postpartum values, but were significantly decreased in second and third trimesters (P less than 0.001). A decrease in TSH levels was observed in the first trimester (0.72 +/- 0.09 vs. 1.23 +/- 0.12 mU/L; P less than 0.001), while second and third trimester values did not differ from those postpartum. A significant negative correlation (P less than 0.05) was observed between hCG and TSH levels in the earliest weeks (8-10) of the first trimester. No correlation was found between hCG and total T4 or free T4 levels. A stimulation of I- uptake in FRTL-5 cells was induced by first trimester serum, which also showed a different behavior at chromatofocusing, with a higher proportion of hCG eluting at acidic pIs compared to second trimester samples. However, neither hCG levels nor the amount of acidic hCG correlated with the thyroid-stimulating activity measured in vitro. Some correlation was found with the percentage of basic hCG (eluting at pI greater than 4.6), although these isoforms were equally present in first and second trimesters. The differing patterns of circulating hCG at various stages of gestation suggest that distinct hCG isoforms may regulate maternal thyroid activity.

Chorionic Gonadotropin↗

PubMed1991

Multianalyte microspot immunoassay--microanalytical "compact disk" of the future.

Throughout the 1970s, controversy centered both on immunoassay "sensitivity" per se and on the relative sensitivities of labeled antibody (Ab) and labeled analyte methods. Our theoretical studies revealed that RIA sensitivities could be surpassed only by the use of very high-specificity nonisotopic labels in "noncompetitive" designs, preferably with monoclonal antibodies. The time-resolved fluorescence methodology known as DELFIA--developed in collaboration with LKB/Wallac--represented the first commercial "ultrasensitive" nonisotopic technique based on these theoretical insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, high-specific-activity labels also permit the development of "multianalyte" immunoassay systems combining ultrasensitivity with the simultaneous measurement of tens, hundreds, or thousands of analytes in a small biological sample. This possibility relies on simple, albeit hitherto-unexploited, physicochemical concepts. The first is that all immunoassays rely on the measurement of Ab occupancy by analyte. The second is that, provided the Ab concentration used is "vanishingly small," fractional Ab occupancy is independent of both Ab concentration and sample volume. This leads to the notion of "ratiometric" immunoassay, involving measurement of the ratio of signals (e.g., fluorescent signals) emitted by two labeled Abs, the first (a "sensor" Ab) deposited as a microspot on a solid support, the second (a "developing" Ab) directed against either occupied or unoccupied binding sites of the sensor Ab. Our preliminary studies of this approach have relied on a dual-channel scanning-laser confocal microscope, permitting microspots of area 100 microns 2 or less to be analyzed, and implying that an array of 10(6) Ab-containing microspots, each directed against a different analyte, could, in principle, be accommodated on an area of 1 cm2. Although measurement of such analyte numbers is unlikely ever to be required, the ability to analyze biological fluids for a wide spectrum of analystes is likely to transform immunodiagnostics in the next decade.

Binding, Competitive↗

PubMed1990

A longitudinal study of total and free thyroid hormones and thyroxine binding globulin during normal puberty.

Thyroid hormones are essential for normal pubertal growth, yet the changes in total and, especially, free thyroid hormones and thyroxine-binding globulin during puberty have not been adequately defined. Serum from 39 normal children (20 girls, 19 boys) between the ages of 10 and 15 years were assayed for total T4, free T4, free T3 and thyroxine-binding globulin at 6-monthly intervals; the free hormone assays were valid, non-analogue methodologies. In the girls, free T4 levels fell from 15.7 +/- 0.6 pmol/l at 10 years to 13.0 +/- 0.6 (p less than 0.001) at 12.5 years before rising to 15.9 +/- 0.7 at 15 years; this nadir occurred at puberty stages 3-4. Changes in total T4 followed a similar pattern with a slight delay in the nadir (13 years, puberty stage 4). In the boys, free T4 fell from 16.3 +/- 0.6 pmol/l at 10 years to 14.3 +/- 0.3 at 13.5 years, then rising to 15.4 +/- 0.5 at 15 years; the nadir again occurred at puberty stages 3-4. The corresponding nadir in total T4 which occurred at puberty stages 4-5 was not apparent by age analysis. Thyroxine-binding globulin concentrations remained unchanged in the girls, but fell slightly in the boys during later puberty. Free T3 concentrations in the girls showed a progressive fall after 12.5 years which was significant by the age of 14 when most had been in puberty stage 5 for more than 1 year. The boys showed no change of free T3 concentration throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1990

Effect of maternal hypothyroxinaemia in the rat on brain biochemistry in adult progeny.

The effects of maternal hypothyroxinaemia during pregnancy on subsequent brain biochemistry in progeny was studied. Normal and partially thyroidectomized rat dams were mated and progeny allowed to grow to adulthood. Brain regions (cerebellum, medulla, midbrain, cerebral cortex and paleocortex) were dissected out and the activities of various cell marker enzymes were determined, along with cholesterol contents. Maternal hypothyroxinaemia was without effect on body weight, brain weight or thyroid status of adult progeny. Oligodendroglial marker enzyme activities were altered in progeny from thyroidectomized dams. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was decreased in the medulla (by 37%) and midbrain (by 32%). 5'-Nucleotidase was also diminished in the same brain regions, by 33% in the medulla and by 35% in the midbrain. In contrast, oleate esterase was increased (by 39%) in the paleocortex. Although these enzymes are putatively involved in myelin metabolism, no changes were observed in the concentration of a major myelin lipid (cholesterol). The activity of beta-D-glucuronidase (a general neuronal marker) was decreased (by 30%) in the paleocortex, whereas N-acetyl-beta-D-galactosaminidase (a general glial marker) was unchanged in all brain regions. In summary, maternal hypothyroxinaemia has irreversible effects on brain biochemistry in adult progeny. The damage is parameter-selective and brain region-specific, analogous to the pattern of neurological damage seen in offspring born to hypothyroxinaemic women in iodine-deficient endemias.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1990

A rat granulosa cell plasminogen activator bioassay for FSH in human serum.

A previously described in-vitro rat granulosa cell plasminogen activator bioassay for FSH has been modified and applied in the assay of human serum. This modified method consists of exposing the diethylstilboestrol-stimulated granulosa cells from 25- to 26-day-old rats to FSH or test substance for 3.5 h in wells coated with 125I-labelled fibrinogen and treated with thrombin. Following stimulation with FSH, the dose-related production of plasminogen activator was measured as the degree of 125I-labelled fibrinolysis in the presence of added plasminogen. Using the urinary FSH/LH bioassay reference preparation as the assay standard, the useful range of the assay was 0.3-15 IU/l, with an assay sensitivity of 0.3 IU/l. As determined using purified glycoprotein hormone preparations, the assay was highly specific for FSH. The minor degree of FSH bioactivity measured in some of the hormone preparations was accounted for by the amount of FSH contamination in these preparations. To abolish interference caused by unknown serum factors, we heat-treated the serum samples for 15 min at 56 degrees C before the assay. The results indicated that neither immunoreactivity nor bioactivity was affected by this treatment. Furthermore, heat-treated human sera gave responses parallel to the standard curve at the three dose levels (2, 4 and 8 microliters) studied. We used this bioassay to estimate the FSH-like bioactivity in 15 human serum samples. The estimates of immunoreactive FSH in these samples correlated well with the corresponding FSH bioactivity (r = 0.745, n = 15 and P less than 0.05). The results indicate that with this sensitive and rapid (completed within 24 h) bioassay, it should be possible to measure FSH bioactivity in heat-treated human serum samples.

Animals↗