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Biomedical subjects

J E Phillips

Publications and source records attributed to J E Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1996

Nature and control of chloride transport in insect absorptive epithelia.

Insect epithelia most commonly absorb from KCI-rich, low Na+ fluids. This is true of the locust hindgut, which is functionally analogous to vertebrate kidney tubules. Active absorption of Cl- at the apical membrane is the predominant transport process giving rise to a large short-circuit current (Isc) after stimulation by neuropeptides (CTSH, ITP) via cAMP as second messenger. This Cl- transport is not coupled to or driven secondarily by Na+, K+, HCO3-, Ca2+, or Mg2+ movements. An apical V-type H+ ATPase acidifies the hindgut lumen but at a rate that is 10-15% of Cl-dependent Isc. The evidence to date as to whether the resulting large apical proton gradient is used to drive Cl- transport secondarily by an apical H+/Cl- symport is mixed. Thus a primary mechanism of Cl- absorption remains an alternative possibility. The complete primary structure of a large neuropeptide stimulant (ITP: 72 amino acids) of locust ileal Cl- transport has recently been deduced from its cDNA. This is the first putative insect neuropeptide hormone shown to stimulate ion transport across absorptive epithelia for which the primary sequence has been deduced.

Amino Acid Sequence↗
PubMed1996

Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system.

Ion transport peptide (ITP) purified from locust nervous corpus cardiacum (CC) has previously been shown to stimulate salt and water reabsorption and inhibit acid secretion in the ileum of Schistocerca gregaria. We used the partial amino acid sequence of purified ITP to derive degenerate primers. These were used to amplify a cDNA from brain RNA using reverse transcription and the polymerase chain reaction (RtPCR). This sequence was extended using anchored PCR to yield a partial, 517bp cDNA clone. This cDNA encodes a putative ITP prohormone which could be cleaved at two dibasic amino acid sites to yield a 72 residue active amidated peptide. The deduced amino acid sequence from the cDNA agrees completely with the amino acid sequence and molecular mass (8564Da) derived from analysis of purified ITP. Relative to a family of crustacean hyperglycaemic hormones (CHH), all six cysteine residues and many other amino acid residues are conserved in ITP, establishing that ITP is a homologue. However, CHH, crab eyestalk and CC extracts from distantly related insects have no action, whereas CC extracts from closely related insects are active on the locust ITP assay, showing that the bioassay is selective. Insect Sf9 cells transfected with a baculovirus containing our partial cDNA secreted a potent stimulant of locust ileal transport, confirming that the peptide encoded by our ITP clone has biological activity. The mRNA for ITP is restricted to the brain and CC. Interestingly, a related mRNA is observed in other tissues which are not active on the ITP bioassay.

Amino Acid Sequence↗
PubMed1995

Marfan syndrome: fibrillin expression and microfibrillar abnormalities in a family with predominant ocular defects.

We have found abnormal fibrillin microfibrils isolated from tissues and cell cultures from two cousins with Marfan syndrome whose major clinical abnormality is bilateral ectopia lentis, but who also have skeletal involvement but no cardiovascular defects. Ultrastructural analysis of ciliary zonules showed the presence of abundant loose microfibril bundles which in many places appeared disorganised. Microfibrils isolated from ciliary zonules and vitreous were highly fragmented when examined by rotary shadowing electron microscopy. Investigation of microfibrils elaborated by patient dermal fibroblasts showed remarkable variations in periodicity and packing. The synthesis and secretion of fibrillin by these cells was confirmed electrophoretically with the identification of metabolically labelled immunoprecipitated fibrillin (M(r) 300,000) in medium and cell layer compartments. These data show that fibrillin expression is normal but that assembled microfibrils are manifestly abnormal both morphologically and functionally. The occurrence of microfibrils with variable periodicities and susceptibility to fragmentation suggests that structural weakness is probably the primary cause of lens dislocation in these patients.

Actin Cytoskeleton↗
PubMed1994

Mutagenesis of recombinant protein C inhibitor reactive site residues alters target proteinase specificity.

Protein C inhibitor (PCI) is a heparin-binding plasma serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C. We are using recombinant PCI (rPCI) to study structural determinants of target proteinase specificity. A cDNA encoding full-length PCI has been expressed as a fully active proteinase inhibitor using Autographa californica nuclear polyhedrosis virus (baculovirus). rPCI was expressed maximally 4 days after infection and could be expressed either in Sf9 or High-Five cells. rPCI bound heparin and was conveniently purified with heparin-Sepharose (eluting > 0.5 M NaCl). The rPCI formed sodium dodecyl sulfate-polyacrylamide gel electrophoresis-stable complexes with thrombin and activated protein C (APC). The inhibitory properties of wild-type rPCI and plasma-derived PCI are essentially the same either in the absence or presence of heparin with thrombin, APC, trypsin, and urokinase. The residues Phe353-Arg354-Ser355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the Arg-Ser peptide bond being cleaved by the proteinase. Using site-directed mutagenesis we studied the contribution of the reactive site FRS for proteinase inhibition in rPCI. Changing the P1 residue Arg354-->Met generated a reactive site similar to alpha 1-proteinase inhibitor which was a much poorer inhibitor of thrombin, APC, trypsin, and urokinase. Changing the P2 residue Phe353-->Gly generated a mutant with a reactive site like antithrombin which was better at inhibiting thrombin or urokinase, but was much less active with APC or trypsin. Changing the P1' residue Ser355-->Met generated a reactive site like plasminogen activator inhibitor-1 and this protein inhibits all the proteinases essentially like wild-type rPCI. These results show the importance of PCI's Phe353 (P2) and Arg354 (P1) in target proteinase specificity, and they further support the concept of reactive site sequences determining serpin function.

Amino Acid Sequence↗
PubMed1994

Fibrillin secretion and microfibril assembly by Marfan dermal fibroblasts.

The Marfan syndrome has been linked to the FBN1 gene encoding the microfibrillar glycoprotein fibrillin. To date, there have been no descriptions of microfibrillar abnormalities characteristic of this connective tissue disorder, although biochemical analyses have highlighted apparent abnormalities in fibrillin synthesis, secretion and processing. We have conducted a biochemical and ultrastructural investigation of fibrillin expression and assembly by a panel of dermal fibroblast lines from patients with Marfan syndrome and related diseases. The study has highlighted marked differences between cells in terms of secretion and aggregation of newly-synthesised fibrillin. In addition, electron microscopic visualization of fibrillin assemblies has clearly demonstrated for the first time the plethora of microfibrillar abnormalities that underlie this heterogeneous disorder. These data emphasize the molecular complexity that is a feature of the diverse clinical phenotypes exhibited by Marfan patients.

Autoradiography↗
PubMed1993

Inhibition of dysthrombins Quick I and II by heparin cofactor II and antithrombin.

Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of glycosaminoglycan. In contrast, glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with glycosaminoglycans and the functional availability of thrombin exosites.

Amino Acid Sequence↗
PubMed1992

Electrogenic proton secretion in the hindgut of the desert locust, Schistocerca gregaria.

The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. Previous studies conducted in the nominal absence of exogenous CO2 and HCO3- suggested that the acidification was due to a proton-secretory rather than bicarbonate-reabsorptive mechanism (Thomson, R.B., Speight, J.D., Phillips, J.E. 1988. J. Insect Physiol. 34:829-837). This conclusion was confirmed in the present study by demonstrating that metabolic CO2 could not contribute sufficient HCO3- to the lumen to account for the rates of rectal acidification observed under the nominally CO2/HCO3(-)-free conditions used in these investigations. Rates of luminal acidification (JH+) were completely unaffected by changes in contraluminal pH, but could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of JH+ was not dependent on Na+, K+, Cl-, Mg2+, or Ca2+ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10-16%) of JH+ measured under these conditions could be attributed to an apical amiloride-inhibitable Na+/H+ exchange mechanism.

Animals↗
PubMed1992

Isolation of a neuropeptide from locust corpus cardiacum which influences ileal transport.

1. Schistocerca gregaria ion-transport peptide (Scg-ITP) was isolated from aqueous extracts of the corpus cardiacum by a four-step procedure, utilizing reverse-phase high-performance liquid chromatography for separation and stimulation of a Cl(-)-dependent short-circuit current (Isc) across locust ilea as the bioassay. 2. Scg-ITP has an unblocked N terminus and an apparent relative molecular mass of 7700. Thirty-one residues (of an estimated 65) were identified by sequence analysis. 3. Scg-ITP is structurally related to a crustacean family of neuropeptides which includes the crustacean hyperglycaemic hormones from the shore crab Carcinus maenas and the crayfish Orconectes limosus and moult-inhibiting hormone and vitellogenesis-inhibiting hormone from the lobster Homarus americanus. 4. Scg-ITP has no sequence homology with neuroparsins (Nps). Nps are the only other neuropeptides isolated to date that might regulate reabsorption in an insect hindgut (rectum).

Amino Acid Sequence↗
PubMed1991

Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides.

We investigated the properties of an artificial chimeric peptide that contains an Arg-Gly-Asp (RGD)-tripeptide, the versatile cell recognition signal of extracellular matrix protein components, coupled to a carboxyl-terminal fragment of the highly specific alpha-thrombin inhibitor, hirudin (residues 53-64): WGRGDSANGDFEEIPEEYL (RGD-hirudin53-64). Hirudin53-64 and RGD-hirudin53-64 inhibited the fibrinogen clotting activity of alpha-thrombin and prolonged the activated partial thromboplastin time of human plasma. In addition, both peptides afforded total protection to thrombin from trypsionolysis. Neither hirudin53-64 nor RGD-hirudin53-64 dramatically interfered with the thrombin-antithrombin inhibition reaction either in the absence or presence of added heparin. alpha-Thrombin-induced platelet aggregation was effectively inhibited by hirudin53-64 and RGD-hirudin53-64. Unlike hirudin53-64, RGD-hirudin53-64 in solution inhibited integrin-mediated endothelial cell and fibroblast cell attachment to polystyrene wells in the presence of fetal bovine serum. Collectively, our results demonstrate that RGD-hirudin53-64 has anticoagulant/antiplatelet aggregation activity attributable to its hirudin sequence and integrin-directed cell attachment activity due to its RGD site. Our results suggest that this chimeric motif may serve as a prototype for a new class of anticoagulants where an integrin-specific sequence "targets" the peptide to a cell (ultimately through the platelet integrin alpha IIb beta 3) trapped amid a thrombus with ensuing proteinase inhibition.

Amino Acid Sequence↗
PubMed1991

Hemolytic activity of ethylene glycol phenyl ether (EGPE) in rabbits.

Studies were conducted to characterize the hemolytic effects of EGPE in rabbits following oral and dermal exposure, and to evaluate the in vitro hemolytic potential of EGPE and its major metabolite using rabbit red blood cells (RBC). Gavage administration of EGPE to female New Zealand White rabbits at 100, 300, 600, or 1000 mg/kg/day for up to 10 consecutive days (one dose/day) resulted in a dose-related intravascular hemolytic anemia. The hemolytic anemia was characterized by decreased RBC count, hemoglobin concentration, packed cell volume, hemoglobinuria, splenic congestion, renal tubule damage, and a regenerative erythroid response in the bone marrow. The hemolytic anemia was observed without alterations in RBC glutathione or methemoglobin. Phenoxyacetic acid (PAA) was identified as a major blood metabolite of EGPE. In vitro exposure of female rabbit erythrocytes indicated EGPE to be considerably more hemolytic than PAA. In a 90-day dermal study in which EGPE was applied to the skin of male and female New Zealand White rabbits 6 hr/day, 5 days/week, at doses up to 500 mg/kg/day, there was no indication of a hemolytic response. The only treatment-related effects were sporadic occurrences of slight erythema and scaling of skin at the site of test material application in high dose group male and female rabbits. However, erythema and scaling were not associated with gross or histopathologic changes and were not considered toxicologically significant.

Anemia, Hemolytic↗
PubMed1991

Effects of various secretagogues on [Ca2+]i in cultured human nasal epithelial cells.

Recent studies conducted on cultured canine tracheal cells have suggested that a complex relationship exists between cAMP and Ca2+ in the control of electrolyte secretion. The goal of this study was to determine if the Ca2+ second messenger system functions in a similar fashion in cultured human nasal epithelial cells, a tissue in which control of electrolyte secretion is known to be disrupted in the genetic disease, cystic fibrosis. Human nasal epithelial tissue was obtained as a by-product of surgery and put into monolayer cell culture. After 4-5 days in culture, cells were loaded with fura-2 and intracellular free Ca2+ measured as previously reported. We found that bradykinin increased intracellular free Ca2+ in all cells tested, whereas isoproterenol increased intracellular free Ca2+ in only half the cells tested, suggesting that more than one transporting cell type may be present in this tissue. Epinephrine and prostaglandin E2 had no effect on intracellular free Ca2+. We found that the voltage-sensitive Ca2+ channel blocker verapamil had no effect on the bradykinin-induced change in intracellular free Ca2+. Removal of Ca2+ from the bathing saline only slightly attenuated the increase in intracellular free Ca2+ that resulted from stimulation with bradykinin. We conclude that the source of the increased intracellular free Ca2+, observed during stimulation with secretagogues, was primarily intracellular stores.

Bradykinin↗
PubMed1990

Stimulants of ileal salt transport in neuroendocrine system of the desert locust.

Proteinaceous factors from the corpora cardiaca (CC) and ventral ganglia (VG) stimulate KCl transport across voltage-clamped preparations of locust ilea in a dose-dependent manner. These factors may act via cAMP as the second messenger because this cyclic nucleotide and agents which increase its cellular level all mimic the stimulatory effects of CC and VG extracts. The CC and VG factors are apparently different compounds because they differ in the time course of ileal short-circuit current (Isc) initiated, in heat stability, and in extraction properties. Because saline extracts of the CC have similar effects on both ileal and rectal segments of locust hindgut, the ileal stimulant from the CC may be the chloride transport-stimulating hormone, described by Phillips et al. (Canad. J. Zool. 58, 1851-1860, 1980) which acts to stimulate rectal Isc.

Animals↗
PubMed1989

Ethyl chloride: 11-day continuous exposure inhalation toxicity study in B6C3F1 mice.

Groups of seven B6C3F1 mice per sex were exposed for 23 hr/day to 0, 250, 1250, or 5000 ppm ethyl chloride (EtCl) for 11 consecutive days to evaluate the potential toxicity of EtCl under near-continuous exposure conditions. On the day following the last exposure, a neurobehavioral observation battery was performed, samples were obtained for clinical chemistry and hematology, and necropsies were conducted. Histopathologic examination was subsequently performed. The only observed effects were increased relative liver weights and a slight increase in hepatocellular vacuolation (glycogen or fat) in 5000 ppm-exposed mice. Exposures to EtCl were well tolerated despite the unusually long exposure periods.

Administration, Inhalation↗
PubMed1988

Undisplaced fracture of the neck of the femur: results of treatment of 100 patients treated by single Watson-Jones nail fixation.

In a review of 100 patients with clinical and radiological evidence of undisplaced Garden stage I and II fractures of the neck of the femur treated by fixation with a single Watson-Jones nail, the early mortality was 4 per cent and in 93 cases with complete radiological data important displacement of the fracture during surgery occurred on one occasion. Of 72 patients with a mean follow-up of 22.6 months (range 3-83 months) 68 patients (94.4 per cent) achieved union and non-union occurred in 4 patients (5.6 per cent) all of whom required further surgery. In 40 patients with united fractures with a minimum follow-up of 2 years (mean 41.2 months; range 24-83 months) the overall incidence of avascular changes was 22.5 per cent; three patients required revisional surgery for late segmental collapse and a further six patients had avascular changes with minimal symptoms. Single nail fixation is a safe reliable procedure and there appears to be little indication for multiple fixation or compression devices for this fracture.

Adult↗
PubMed1988

Some major transport mechanisms of insect absorptive epithelia.

1. After hormonal stimulation, fluid reabsorption (JV) in locust hindgut from the KCl-rich, low-Na primary urine is driven primarily by an unusual mucosal electrogenic Cl- pump (JCl). 2. Cyclic-AMP increases JCl and also mucosal K+ and basolateral Cl- conductances, so that KCl absorption exceeds that of Na+ in rectum but not ileum. 3. Mucosal entry of Na+ occurs by exchange for NH4+ (H+), by cotransport with some neutral amino acids, and through putative channels. 4. However, transport of proline, the predominant organic substrate, is largely Na-independent and drives a sizable component of Jv in rectal but not ileal segments. 5. There is evidence for hormonal control of Na+ reabsorption in ileum but not rectum.

Absorption↗
PubMed1988

NH4+ transport in acid-secreting insect epithelium.

The relationship between ammonia secretion (JAmm), hydrogen ion secretion (JH), and intracellular pH (pHi) was investigated in isolated locust (Schistocerca gregaria) rectal epithelia mounted as flat sheets in specially designed Ussing chambers. In the absence of exogenous CO2 and ammonia, the rectum is capable of net acid and ammonia secretion into the lumen against pH gradients of up to 1.8 U. JAmm was dependent on the presence of luminal amino acids and Na+ and it was relatively unaffected by K+ removal or changes in membrane potential. JAmm and pHi remained fairly constant over a luminal pH range of 7-5, whereas JH decreased linearly to zero over the same pH range. Mucosal addition of 1 mM amiloride reduced JAmm by 60%. This study demonstrates that the locust rectum secretes significant quantities of endogenously produced ammonia preferentially into the lumen as NH+4 rather than NH3. Moreover, the results suggest that the ammonia crosses the apical membrane via an amiloride-inhibitable Na+-NH+4 exchange mechanism.

Amiloride↗