Biomedical subjects

P J Phizackerley

Publications and source records attributed to P J Phizackerley.

At least 19 recordsLinked to original sources

PubMed2000

Fetal surfactant as a source of arachidonate in human amniotic fluid.

The factors responsible for the onset of labor in women are not well understood but it is clear that parturition is associated with increased production of prostanoids and release of arachidonic acid by intrauterine tissues. Pulmonary surfactant is secreted from the fetal lung into the amniotic fluid where its concentration increases toward term. In this paper we have shown that the ability of fetal surfactant to stimulate prostaglandin production by amnion cells is greatly enhanced by pre-incubating surfactant with amniotic fluid. This is due to the release of fatty acids, including arachidonate, from the lipids of fetal surfactant by the sequential action of phospholipase C and diglyceride lipase. Thus, in addition to providing the amnion with a source of arachidonate derived from the intracellular transfer of arachidonate from surfactant phosphatidylcholine to phosphatidylethanolamine and phosphatidylinositol in amnion cells, fetal surfactant also contributes to the pool of free arachidonate in amniotic fluid.

Amniotic Fluid↗

PubMed1994

Surfactant proteins A (SP-A) and D (SP-D): levels in human amniotic fluid and localization in the fetal membranes.

Surfactant proteins A (SP-A) and D (SP-D) are major proteins, in the lung, which are composed of collagenous and globular domains. They show an overall similarity to the serum complement protein Clq, which is involved in the initiation of antibody-dependent defence mechanisms. Both SP-A and SP-D were detected, immunochemically, in amniotic fluid as early as 26 weeks gestation and, as expected, SP-A levels rose sharply from 32 weeks towards term. By contrast, SP-D levels in the same samples rose only moderately. Immunochemistry of paraffin sections of fetal membranes, revealed the presence of both SP-A and SP-D in the amniotic epithelium and chorio-decidual layers. SP-A and SP-D are both lectins and therefore they may play a role in the antibody-independent recognition and clearance of pathogens in the amniotic fluid.

Amniotic Fluid↗

PubMed1993

Utilization by human amniocytes for prostaglandin synthesis of [1-14C]arachidonate derived from 2-[1-14C]arachidonylphosphatidylcholine associated with human fetal pulmonary surfactant.

The phospholipids of human fetal pulmonary surfactant prepared from term amniotic fluid contained arachidonic acid and its utilization for prostaglandin synthesis by amnion cells has been investigated. Cells were incubated with surfactant labelled with L-alpha-1-palmitoyl-2-[1-14C]arachidonylphosphatidylcholine. The uptake of radioactivity into amniocyte phospholipids increased with time and with the concentration of surfactant and after 2 h of incubation at 37 degrees C, 63% of the incorporated radioactivity was recovered in phosphatidylethanolamine (PE) and phosphatidylinositol (PI). Similar results were obtained when amniocytes were incubated with liposomes prepared from lipid extracts of surfactant, but when cells were incubated with liposomes prepared from synthetic lipids the transfer of radioactivity to PE and PI was only 27%. Fetal surfactant contained platelet activating factor (PAF) but the addition of the antagonist hexanolamino-PAF did not affect either the uptake or intracellular redistribution of surfactant arachidonate by amniocytes, nor did the addition of PAF affect the results obtained with liposomes prepared with synthetic lipids. Cells preincubated with surfactant labelled with 2-[1-14]arachidonylPC released radioactive arachidonate and prostaglandin E2 when stimulated with calcium ionophore A23187 or with phorbol ester. These data demonstrate that surfactant provides a source of arachidonate that can be utilized by amnion cells for prostaglandin synthesis.

Amnion↗

PubMed1992

Platelet-activating factor levels in human follicular and amniotic fluids.

Platelet-activating factor (PAF) was estimated in extracts of human follicular and amniotic fluids using a commercially available 125I-radioimmunoassay. Levels obtained before and after purification of the extracts by thin-layer chromatography were similar and PAF could be estimated with high accuracy and reproducibility over a wide dilution range. PAF levels in fluid aspirated from mature (17 mm average diameter) follicles from in vitro fertilization patients were 1005 +/- 129 fmol/ml in successful (clinical pregnancy) cycles and 949 +/- 75 fmol/ml in unsuccessful (failure of implantation) cycles. PAF levels were also similar in follicles containing eggs and those where no egg was found, and in fluid from follicular cysts characterised by low oestradiol and progesterone levels. PAF was detected in midtrimester amniotic fluid (60 +/- 20 fmol/ml). At term, PAF was low or undetectable in amniotic fluid obtained at elective caesarean section before the onset of labour, but increased to 102 +/- 28 fmol/ml in samples obtained following spontaneous labour and vaginal delivery. The data suggest that PAF may be involved in ovulation and parturition but further studies into the mechanism of action of PAF are necessary. The good performance of the radioimmunoassay and the convenience of gamma-counting are strong advantages of this method over the available bioassays, but its expense remains a drawback.

Amniotic Fluid↗

PubMed1992

Human placental phospholipase A2 activity in term and preterm labour.

Phospholipase A2 activity (EC 3.1.1.4) was estimated in low-speed supernatants of human placenta by measuring the release of arachidonic acid from phosphatidylcholine, 1-stearoyl-2-[3H]arachidonyl and other phospholipids under alkaline conditions (pH 8). Activity was calcium dependent and followed Michaelis-Menten kinetics with an apparent Km of 0.19 (SD 0.04) mM and a Vmax of 486 (SD 52) pmol/mg protein per h. Placental phospholipase A2 activity was relatively high in early pregnancy (8-11 weeks gestation) and following spontaneous preterm labour (31-34 weeks), but decreased significantly towards term with no changes in relation to parturition. By contrast, activity in the fetal membranes (amnion and chorio-decidua) was low after preterm labour and increased significantly towards term. The placenta and fetal membranes have the capacity to contribute to the increased intra-uterine release of arachidonic acid and eicosanoids that occurs during term and preterm labour, but at present there is no evidence that this increase involves direct changes in phospholipase A2 activity.

Amnion↗

PubMed1991

Adsorption of fetal surfactant protein SP-B on the human amnion at term and on amniocytes incubated with fetal surfactant in vitro.

Fetal surfactant stimulates the synthesis of prostaglandins by slices of human amnion at term and by a human amnion cell line, and these effects are partly dependent upon surfactant apoproteins. In this paper, methods are described for the purification of surfactant from human amniotic fluid and from post-mortem human lung. A procedure is described for the purification of surfactant protein SP-B from human amniotic fluid, and the sequence of 20 amino acids at the N-terminal has been determined. A monoclonal antibody generated against human lung surfactant has been shown to react with SP-B from amniotic-fluid surfactant, and the presence of SP-B on the surface of the amnion at term has been demonstrated by immunohistochemical methods. It has also been shown that SP-B from surfactant is present on the surface of amniocytes incubated with surfactant in vitro.

Adsorption↗

PubMed1989

Effect of lipid and protein fractions from fetal pulmonary surfactant on prostaglandin E production by a human amnion cell line.

Discs of amnion and choriodecidua prepared from women delivered at term were incubated with and without surfactant prepared from human amniotic fluid and the output of prostaglandin E (PGE) was estimated by radioimmunoassay. Surfactant stimulated the release of PGE in both tissues. The stimulatory effect was characterized further using cultured human amnion cells. The effect was dose- and time-dependent and required for full expression both protein and lipid components of surfactant. Arachidonate constituted about 3.5% of the fatty acids of fetal surfactant phospholipids. These findings suggest that surfactant is an important source of arachidonate for intrauterine PG synthesis.

Amnion↗

PubMed1988

Surfactant stimulates prostaglandin E production in human amnion.

Discs of human amnion prepared from nine women delivered by elective caesarean section at term were incubated with and without purified human amniotic fluid surfactant (9 micrograms lipid P/ml), and the output of prostaglandin E (PGE) was estimated by radioimmunoassay. Surfactant stimulated the release of PGE from 3.8 (SD 2.9) to 5.4 (SD 2.5) pmol/mg dry weight/3 h (P less than 0.01). Arachidonic acid accounted for 2% of the fatty acids of surfactant lecithin. These results show that surfactant has a stimulatory effect on amniotic PGE production and that it is an important source of arachidonic acid in amniotic fluid.

Amnion↗

PubMed1986

Effect of ischemia and reperfusion of pig skin flaps on epidermal glycogen metabolism.

Pedicled skin flaps in the pig have been used to investigate the effects of 3-h ischemia and reperfusion on the epidermal metabolism of glycogen and glucose. Epidermal glycogen content fell steadily at a rate of about 1.2 mumol of glucose-equivalents per g wet weight per h whereas the rate of glucose consumption declined from 1.8 mumol per g wet weight during the first hour to about 0.25 mumol per g wet weight in the third hour. During ischemia the proportion of glycogen synthase in the I form increased progressively from an initial value of about 8% to about 70%, but the proportion of phosphorylase in the a form decreased only in the third hour of ischemia. The concentration of ATP decreased and ADP and AMP increased but the total pool of epidermal adenine nucleotides was not depleted. On reperfusion, these changes were reversed and normal epidermal concentrations of glucose and adenine nucleotides were restored within 30 min and remained stable thereafter. The resynthesis of glycogen proceeded at a steady rate of about 1 mumol per h per g wet weight and the phosphorylation state of both glycogen synthase and phosphorylase approached normal values after 3 h. It is concluded that epidermal glycogenolysis in ischemia is, at least in part, a consequence of activation of phosphorylase b by AMP, and that glycogen resynthesis on reperfusion is promoted by the ischemic activation of glycogen synthase.

Adenine Nucleotides↗

PubMed1984

Glycogen metabolism in psoriatic epidermis and in regenerating epidermis.

The observation that the glycogen content of epidermis from psoriatic lesions and from regenerating wound epithelium is increased has been confirmed by quantitative estimation. In epidermis from psoriatic lesions, although the proportion of glycogen synthase in the I form is only about 5% of the total and similar to control values, total glycogen synthase activity is increased approximately 4-fold and hence glycogen synthase I activity is increased to the same extent. In contrast, total phosphorylase activity is only slightly increased and, since the proportion of the enzyme in the a form is reduced, phosphorylase a activity is similar to control values. In epidermis from psoriatic lesions, the concentration of UDP-glucose is approximately doubled, and the concentrations of fructose 1,6-bisphosphate and of 6-phosphogluconate are increased approximately 5-fold. It is concluded that rates of glycogen synthesis, of glycolysis and of the pentose phosphate pathway are all enhanced in vivo and in consequence the rate of glucose uptake by psoriatic epidermis must be increased. In the non-involved epidermis of psoriatic patients the glycogen content is within normal limits, and although total glycogen synthase activity is increased the ratio of glycogen synthase I to phosphorylase a is maintained at normal levels by the appropriate phosphorylation of both enzymes. In regenerating wound epithelium in the pig, the changes in enzyme activity and in metabolite concentration closely resemble those found in epithelium from psoriatic lesions except that in wound epithelium the proportion of phosphorylase in the a form is increased relative to normal epithelium.

Animals↗

PubMed1983

The effects of starvation and re-feeding on glycogen metabolism in mouse tail skin.

Although the glycogen content of mouse tail skin was decreased during starvation and was restored on re feeding, the proportion of glycogen synthase in the I form remained constant throughout at about 10% of the total. During the phase of net glycogen synthesis 1.5h after access to food was restored, the concentration of UDP glucose was markedly increased and the proportion of phosphorylase in the a form was significantly decreased.

Animals↗

PubMed1983

The reversible detachment and deposition of surfactant and of pulmonary macrophages during bronchopulmonary lavage in the rat.

A procedure is described for carrying out repetitive bronchopulmonary lavage in the rat, in which a given volume of lavage fluid is introduced into the lungs from a reservoir and then withdrawn from the lungs back into the reservoir, the process being repeated a number of times. During this procedure there is a net release of endogenous surfactant and macrophages from the lungs. [14C] pulmonary surfactant was prepared from rats previously injected intravenously with [1-14C] palmitate, and pulmonary macrophages labelled with 85Sr were prepared from rats which had received by intratracheal injection a suspension of fused clay particles labelled with 85Sr. It was shown by carrying out repetitive bronchopulmonary lavage with 0.15M-NaCl containing either exogenous [14C]-surfactant or [85Sr]-macrophages that the release of endogenous surfactant and macrophages from the lung into the lavage fluid occurred concomitantly with the retention within the lung of radioactive exogenous surfactant and macrophages from the lavage fluid. It is concluded that the process of surfactant and macrophage detachment during bronchopulmonary lavage is reversible and that the exchange processes are of substantial magnitude.

Animals↗

PubMed1983

The estimation of nitrate and nitrite in saliva and urine.

A method for the estimation of nitrate and nitrite is described in which nitrate is converted to nitrite by Klebsiella pneumoniae (UNF 9232) and nitrite is estimated by the Griess reaction before and after incubation. The method is suitable for the estimation of 1-25 nmol of each ion in body fluids, many samples can be handled simultaneously, and special apparatus is not required.

Humans↗

PubMed1983

Glucose metabolism in experimental skin flaps.

The metabolism of glucose by rat abdominal skin flaps has been investigated at various times after flap elevation. Biopsies of flap skin taken during the first 3 days after flap elevation and incubated in vitro show a marked increase in glucose consumption and lactate production compared with normal skin. At the same time, flap tissue reserves of glucose and glycogen are lower than those of normal skin. Providing that some circulation persists, the magnitude of the changes in glucose metabolism is proportional to the degree of ischemia experienced by the flap tissue in vivo. In most cases, glucose consumption and lactate production return to normal by the end of the first week after flap elevation. These experiments thus further define a major ischemia-induced shift to anaerobic metabolism (glycolysis) that occurs in skin flaps.

Animals↗

PubMed1979

Hydrophobic proteins of lamellated osmiophilic bodies isolated from pig lung.

1. In addition to proteins that are insoluble in organic solvents, lamellated bodies isolated from pig lung and surfactant prepared from bronchopulmonary lavage fluid contain another group of proteins that are extracted together with lipid into the organic phase. 2. These hydrophobic proteins constitute about 40% of the total protein of lamellated bodies and about 13% of the total protein of surfactant isolated from lavage fluid, whereas less than 1% of the total protein of pig lung microsomal fraction and mitochondria is extracted by organic solvents. 3. The hydrophobic proteins of lamellated bodies were separated into four fractions and freed from phospholipid by chromatographic procedures. Their apparent molecular weights vary between 11 500 and 16 500, they contain 72--79% of hydrophobic residues and 16--22% of sulphur-containing amino-acids, and leucine is the major N-terminal amino acid in each case.

Amino Acids↗

PubMed1979

Structural studies on lamellated osmiophilic bodies isolated from pig lung. 31P NMR results and water content.

1. Lamellated osmiophilic bodies are intracellular organelles in which pulmonary surfactant is stored prior to secretion. They contain about 85% phospholipid (per dry weight) and dipalmitoyl phosphatidylcholine is a major constituent, and although their ultrastructure is uncertain it is generally supposed that they resemble liposomes. However, liposomes are stable because layers of water are interposed between the lipid bilayers whereas an essential aspect of the function of lamellated bodies is that, subsequent to their secretion, they are rapidly disrupted to form a surface-active film which covers the respiratory epithelium of the lung. 2. A new method for isolating lamellated bodies from pig lung is described which has the advantage of speed and simplicity and which results in increased yields. The homogeneity of the preparation is similar to that obtained by other methods. 3. 31P NMR spectra of lamellated bodies showed that at 40 degrees C about 95% of the phospholipid was present as extended bilayers and that about 5% was present in a phase exhibiting isotropic head group mobility (tau R less than 10(-5) s). It is suggested that this phase may be due to apolar proteins which are present both in lamellated bodies and in liposomes prepared from lipids extracted from them. 4. The internal water content of lamellated bodies has been measured gravimetrically and the hydration of the phospholipid head groups has been examined by 31P NMR. The two methods gave results in good agreement and show that there are about seven molecules of water/molecule of phospholipid. It is concluded that although the phospholipid head groups in lamellated bodies are fully hydrated, there is no zone of free water. 5. Lamellated bodies are osmotically insensitive to NaCl whereas liposomes prepared from lipids extracted from them behave like perfect osmometers. It is suggested that the osmotic insensitivity and restricted water content of lamellated bodies are important to their function and dependent upon polar proteins in the outer limiting membrane.

Animals↗

PubMed1979

The kinetics of macrophage and surfactant replacement in hamster lung following bronchopulmonary lavage.

The effects of bronchopulmonary lavage with isotonic NaCl have been investigated in the hamster, and methods are described for separating pulmonary surfactant from cells in lavage fluid and for the quantitative isolation of surfactant from lung. It has been shown that hamsters almost invariably survived a lavage which consisted of ten successive washes each of 2 ml saline. This procedure removed about 80 per cent. of the total surfactant originally present and the surfactant content of the lung was restored to normal within 24 hr. The same procedure however removed at most only about 25 per cent. of the total number of macrophages in the lung and although a second lavage performed immediately after the first removed large numbers of macrophages, if a delay of several hours elapsed between the two lavages, the yield of macrophages was greatly reduced. The reasons for this behaviour are not understood, and it is likely to be one of the factors which limits the frequency with which pulmonary lavage can be repeated.

Animals↗

PubMed1976

Glucose-6-phosphate dehydrogenase in lichen planus skin.

The apparent Michaelis constant (Km) for glucose-6-phosphate of the enzyme glucose-6-phosphate dehydrogenase has been measured in extracts prepared from biopsies of normal human skin and from both affected and apparently normal skin of patients with lichen planus. No differences of Km were found and starch gel electrophoresis of extracts from lichen planus lesions and normal controls showed similar patterns when stained for glucose-6-phosphate dehydrogenase activity. These results do not support the view that lichen planus is an inborn error of metabolism in which the structure of glucose-6-phosphate dehydrogenase of skin is affected.

Electrophoresis, Starch Gel↗