Search PubMed⌕ Search

Biomedical subjects

W Gu

Publications and source records attributed to W Gu.

142 records · Page 8Linked to original sources
PubMed1989

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains.

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

Biopolymers↗
PubMed1989

Production of corticotrophin releasing hormone by the sheep placenta in vivo.

Corticotrophin-releasing hormone (CRH)-like activity has been reported in placental tissue and to rise sharply in maternal and fetal plasma during the third trimester of human pregnancy. It is unclear whether this applies to other species, if the placental secretes CRH, and if so what factors regulate its production. The present experiments were conducted on sheep 123-144 days pregnant. CRH-like activity was detected in the plasma of the uterine and umbilical vein at modest concentrations. These concentrations rose in the final days before delivery. Reduction of uterine blood flow, particularly caused by an elevation of maternal adrenaline, had the capacity to sharply increase placental output. The CRH-like activity on separation by hplc had the characteristics of 41CRH. The results are discussed in relation to the potential role of placentally-derived CRH.

Animals↗
PubMed1988

Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function.

To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.

Animals↗
PubMed1988

Cerebral blood flow in the fetal guinea-pig.

To measure brain blood flow in the fetal guinea-pig, radioactive microspheres were injected in the lateral saphenous vein whilst a reference sample of blood was withdrawn from the right axillary artery. Measurements were made near term of pregnancy, on the 60th-66th day, during anaesthesia with pentobarbitone and diazepam. Fetal blood pressure was 4.25 +/- 0.12 kPa and fetal heart rate was 250 +/- 7 beats per min. The arterial oxygen content varied between 1.9-5.1 mmol 1(-1). Blood flow to the whole brain (mean 1.13 +/- 0.14 ml min-1 g-1) was significantly correlated to the reciprocal of arterial oxygen content (r = 0.84). Four regions of the brain were examined: the cerebral hemispheres, the cerebellum, the thalamus and midbrain, and the pons and medulla. In each region blood flow was inversely related to arterial oxygen content (r = 0.80-0.83) but the rate of perfusion of the brain stem was greater than that of the cerebral hemispheres or cerebellum.

Animals↗
PubMed1988

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II.

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.

Animals↗
PubMed1987

Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules.

Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells.

Animals↗
PubMed1987

Metabolism of glucose by fetus and placenta of sheep. The effects of normal fluctuations in uterine blood flow.

The metabolism by the fetus and placenta of [2-3H, U-14C]glucose infused into fetal sheep has been studied. Uptake of glucose from the fetus by the placenta and transfer to the ewe, as well as placental metabolism of glucose to fructose and lactate have been quantified. About two-thirds of the glucose removed from the fetal circulation was taken up by placenta. Less than 15% of this passed back into the maternal circulation, the remainder was converted, at roughly equivalent rates, into lactate and fructose, most of which was transferred back to the fetus. It seems likely that little of this glucose is oxidised by the placenta. This data indicates that there are substrate cycles between the placenta and fetus, one possible function of which is to limit fetal glucose loss back to the mother; lactate and fructose have limited placental permeability. At uterine blood flow rates in the middle of the normal range net glucose uptake by the placenta from the maternal circulation was about 7-fold higher than that from the fetus. About 20% of this was transported to the fetus, 50% was oxidised and much of the remainder converted to lactate and transferred back to the ewe. Labelling patterns in fructose and lactate make it unlikely that this placental pool of glucose mixes freely with that derived from uptake from the fetus. Net movement of glucose across the placenta is markedly influenced by fluctuations in uterine blood flow over the normal range of 500-3000 ml/min. At low flow rates there is net output of glucose from the fetus to the placenta, and in some instances from the placenta to the ewe, i.e. there is evidence of net utero-placental production of glucose to the ewe separate from output by the fetus. There is a close linear relationship between uterine glucose supply (maternal arterial concentration x uterine blood flow) and net balance across the placenta. As uterine supply of glucose falls there is increased uptake by the placenta of glucose from the fetal circulation and corresponding enhanced recycling of fructose and lactate to the fetus. This production of fructose and lactate by the placenta may function to reduce glucose loss from the fetus to the ewe. Hence at high rates of placental uptake of glucose from the fetus placental production of lactate and particularly fructose may approach saturation and allow significant backflow of glucose from the fetus to the ewe. Under these conditions glucose uptake may in part sustain placental oxygen consumption.

Animals↗
PubMed1986

The effect of elevation of maternal plasma catecholamines on the fetus and placenta of the pregnant sheep.

To study the effects of reduced uterine blood flow on fetal and placental metabolism, adrenaline has been infused at physiological doses (0.5 microgram/min per kg) into the circulation of the pregnant sheep. This gives a reduction of about one third of uterine blood flow at days 120-143 of pregnancy, but causes no significant change in umbilical blood flow. In contrast to the effects of constricting the uterine artery to reduce blood flow to a similar degree, placental oxygen consumption was reduced and that, together with a large increase in lactate production, indicated the placenta became hypoxic. The fetal blood gas status and hence oxygen consumption was not affected significantly. A consistent arterio-venous difference for glucose across the umbilical or uterine circulations was not detected unless the uterine blood flow was comparatively high. Glucose balance across the uterus showed a close linear relationship with uterine blood flow and more particularly with the supply of glucose to the uterus. There was clear evidence for glucose uptake by the placenta and fetus and also glucose output by both. The latter was more common when uterine blood flow was comparatively low or reduced by adrenaline infusion. The results are consistent with the concept that glucose supply has to be maintained to the placenta even at the expense of fetal stores, although lactate can substitute if there is enhanced output because of fetal hypoxia. They indicate that placental mobilisation of glycogen can lead to a net output of glucose to the mother. The manner of communicating to the fetus changes in placental state that occur during maternal adrenaline infusion is not clear. However towards the end of the 60 min infusion, elevation of fetal plasma adrenaline, probably resulting from a breakdown of the placental permeability barrier, may be an important signal.

Animals↗
PubMed1985

Metabolic and cardiovascular effects on fetal sheep of sustained reduction of uterine blood flow.

The effects on the fetus and placenta of graded reductions of uterine blood flow to 30-90% of control have been studied in sheep at days 125-143 of pregnancy. Reduction of uterine flow to 70-90% of control had little effect upon fetal oxygenation or heart rate or blood pressure but elevated fetal plasma catecholamine concentration. Reduction of flow to 30-50% of control depressed fetal arterial and umbilical venous PO2 but had little effect upon oxygen consumption unless the umbilical venous value fell below about 14 mmHg when it was depressed by up to 30%. Placental oxygen consumption did not fall and was therefore maintained at the expense of the fetus. Fetal arterial pressure rose by 10-12 mmHg and heart rate fell by about 30 beats/min during the first 10-15 min then rose above its initial value. Plasma adrenaline and noradrenaline concentrations rose progressively at a rate which increased with greater degrees of asphyxia. When uterine blood flow was reduced below one-half of normal, net placental consumption of glucose fell and there was evidence of substantial provision of glucose and lactate from the fetus. Fetal production of lactate increased sharply and much of this appeared to be consumed by the placenta at a rate sufficient to account entirely for the deficit in net glucose consumption. The results are consistent with the hypothesis that the fetus senses even small changes in uterine blood flow that are alone insufficient to elicit significant blood gas changes. When the fall in uterine flow caused by arterial compression is relatively large, nutrient supply to the placenta is maintained at the expense of the fetus and as a result of fetal glucose and lactate production. The elevation of fetal arterial PCO2 appears to enhance fetal responses to hypoxia. The results are discussed in relation to the fetal responses to brief and prolonged reductions in uterine blood flow.

Animals↗
PubMedDate not supplied

Characterisation of soy isoflavones and screening for novel malonyl glycosides using high-performance liquid chromatography-electrospray ionisation-mass spectrometry.

HPLC combined with electrospray ionisation (ESI)-MS and photodiode array detection has been employed to study the isoflavone components of soy. All of the known soy isoflavones separated by HPLC were identified and characterised, and three novel isoflavones were detected and screened out. These minor isoflavones were deduced to be isomers of 6"-O-malonyl isoflavone glycosides, based on the ESI-MS and UV data, in which the malonyl group is attached at a position other than the 6" position of the glycosyl moiety of the molecule. These novel malonyl glycosides are as thermally labile as the 6"-O-malonyl glycosides, being converted into known isoflavone glycosides after heating in aqueous ethanol. The advantages of HPLC-ESI-MS in detection of novel isoflavones from plant extracts are reviewed.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Individual DNA bands obtained by RAPD analysis of canine genomic DNA often contain multiple DNA sequences.

The random amplified polymorphic DNA (RAPD) technique has been widely applied for genetic studies of plants, insects, and fungi, and recently has been used for studies in animals including dogs. To convert the RAPD marker into a classical PCR marker, the RAPD-PCR products are size-separated in an agarose gel and a specific DNA band is selected for potential association with a trait of interest. The DNA fragments present in the desired band are then cloned and sequenced using primers specific to the cloning vector, and the sequence is used to design a pair of classical PCR primers. Often a "positive clone" is identified based solely on a match of the size of the insert in the clone with the uncharacterized DNA band originally selected. We observed that single DNA bands obtained from RAPD-PCR using canine genomic DNA often contain DNA fragments of similar size but of different sequences. Based on this observation, we report here a modification of the protocol for RAPD analysis which will ensure that a promising RAPD marker selected based on initial screening is not lost for lack of a comprehensive investigation in the later experimental analysis.

Animals↗
PubMedDate not supplied

Developmental expression of glutathione peroxidase, catalase, and manganese superoxide dismutase mRNAs during spermatogenesis in the mouse.

We have examined in mouse testis the steady-state levels of mRNAs encoding glutathione peroxidase (GSHPx), catalase (CAT), and superoxide dismutase 2 (SOD-2), three enzymes essential for the antioxidant protection of cells. In RNA preparations derived from prepuberal and adult testes and from isolated populations of meiotic and post-meiotic germ cells, one major GSHPx mRNA of about 0.8 kilobases (kb) and one major CAT mRNA of about 2.4 kb were detected. Three SOD-2 mRNAs of about 2.2, 1.2, and 1.0 kb were found in testis. In contrast to GSHPx and CAT, the mRNA levels of SOD-2 were higher in testis than in liver. SOD-2 mRNA levels are developmentally and translationally regulated with maximal levels of expression in early post-meiotic germ cells, whereas the levels of GSHPx and CAT mRNAs are relatively constant in both prepuberal and adult testes. These data suggest that translational regulation plays a more prominent role for SOD-2 expression than for GSHPx or CAT expression in the mammalian testis.

Animals↗
PubMedDate not supplied

Sertoli cells in culture and mRNA differential display provide a sensitive early warning assay system to detect changes induced by xenobiotics.

We have used cultured rat Sertoli cells as an "early warning system" to monitor for morphological and biochemical changes induced by two different xenobiotics-cadmium acetate and polychlorinated biphenyls (PCBs). Sertoli cells begin to round, become vacuolized, and detach from their substrate within 24 hours of culture in the presence of cadmium at concentrations of 0.5-1.0 microM. Similar results were obtained with a lower dose of cadmium (0.01 microM) after 72 hours. When Sertoli cells are cultured for 24 hours in the presence of a mixture of PCBs (3,3',4,4'-tetrachlorobiphenyl, 2,2',4,6,6'-pentachlorophenyl, and 2,2',3,3',4,5,5', 6,6'-nonachlorobiphenyl) at concentrations of 1.0-2.0 microM, they enlarge. After 72 hours, a lower dose of PCBs (0.01 microM) produces similar cellular enlargement. Despite their changes in morphology, no reduction in Sertoli cell viability was seen at any of the concentrations or time points studied for either toxicant. Using mRNA differential display, a number of novel cDNAs were detected when cells were cultured with either cadmium or the PCBs, demonstrating that changes in gene expression accompany the changes in Sertoli cell structure. We propose that Sertoli cells in culture and mRNA differential display provide a sensitive morphological and biochemical assay system to detect early direct effects of low concentrations of toxicants on mammalian Sertoli cells.

Acetates↗