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Biomedical subjects

G J Phillips

Publications and source records attributed to G J Phillips.

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

Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle.

Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep-inv) that inserts with an inverted topology compared to the wild-type protein was studied in Escherichia coli strains that are conditional for the expression of either Ffh or 4.5S RNA, the two components of the E. coli SRP. Depletion of either component strongly affected the insertion of both Lep and Lep-inv into the cytoplasmic membrane. This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E. coli.

Escherichia coli↗
PubMed1996

Sulphur dioxide: a potent glutathione depleting agent.

Sulphur dioxide (SO2) is an air pollutant implicated in the initiation of asthmatic symptoms. Glutathione (GSH) has been proposed to play a role in detoxification of SO2 through the sulfitolysis of glutathione disulphide (GSSG) to S-sulphoglutathione (GSSO3-). Rats were exposed to concentrations of SO2 between 5 and 100 ppm for 5 hr a day between 7 and 28 days. Lung injury as assessed by bronchoalveolar lavage and tissue GSH status were evaluated. SO2 5 ppm failed to elicit any lung injury or inflammatory response but did deplete GSH pools in lung, liver, heart and kidney. Activities of gamma-glutamylcysteine synthetase (GCS), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GRed) in lung were lowered relative to those in control animals. In liver, GRed activity was decreased. SO2 50 ppm exposure also failed to elicit injury or inflammation but did lower inflammatory cell numbers in the circulation. Rats exposed to 50 ppm SO2 maintained tissue GSH status, but activities of GCS, GPx, GRed and gamma-glutamyltranspeptidase in lung and hepatic GRed and GPx were significantly lower than in control rats. Unaltered GST activity in lung and liver was suggestive of an impairment of the sulfitolysis reaction in these animals, perhaps through lower substrate flux through the GPx reaction, as GSSO3- is a known inhibitor of GST in the rat. Rats exposed to 100 ppm SO2 exhibited evidence of inflammation (120-fold increase in neutrophil numbers recovered in lavage fluid) and like the 5 ppm exposed rats had lower tissue GSH concentrations and GSH-related enzyme activities in lung. We conclude that sulfitolysis of GSSG does occur in vivo during SO2 exposure and that SO2, even in the absence of pulmonary injury, is a potent glutathione depleting agent.

Air Pollutants↗
PubMed1995

New plasmids carrying antibiotic-resistance cassettes.

A series of new plasmid vectors is described that carry gene cassettes imparting resistance to the antibiotics chloramphenicol (CmR), kanamycin (KmR), tetracycline (TcR) and spectinomycin/streptomycin (Sp/SmR). The gene cassettes are symmetrically flanked by several restriction sites. In addition, several restriction sites that are normally found internal to the gene cassettes have been eliminated, thereby expanding the number of restriction enzymes available to excise an intact antibiotic-resistance gene. The gene cassettes are carried by high-copy-number plasmids that confer ampicillin resistance (ApR).

DNA Transposable Elements↗
PubMed1995

Oxygen-induced lung injury in the pre-term guinea pig: the role of leukotriene B4.

Leukotriene B4 (LTB4) has been reported to promote the formation of lung oedema when infused into the pulmonary circulation of adult animals. The present study evaluated the hypothesis that LTB4 was responsible, in part, for the oedema that develops during oxidative injury of the immature lung. Significant increases were found in LTB4 concentration in bronchoalveolar lavage fluid obtained from pre-term guinea pig pups maintained in 95% oxygen for 48 h (P < 0.05) and 72 h (P < 0.05) compared to pups maintained in 21% oxygen. Cellular analysis of lavage fluid revealed a concurrent influx of neutrophils into the hyperoxic-injured lung at these times. The protein concentration of lavage fluid was also increased by 48-h hyperoxia exposure indicating elevated pulmonary microvascular permeability. In a second series of experiments, pups exposed to 95% oxygen (and 21% oxygen controls) were treated with a specific LTB4 antagonist (U-75302), at either 0.5, 1.5 or 3.0 mg 100 g body wt to ascertain if LTB4 played a role in either neutrophil recruitment or oedema formation in the immature lung. The number of neutrophils recovered in bronchoalveolar lavage fluid was significantly reduced, compared to vehicle-treated pups, in pups treated with U-75302, at both 1.5 and 3.0 mg/100 g body wt but not 0.5 mg/100 g body wt. Histopathological analysis of pups treated with 1.5 mg U-75302/100 g body wt revealed fewer neutrophils in the pulmonary interstitium (198 vs. 218 mm-2, P < 0.05). The extent of lung microvascular permeability, elevated by hyperoxic exposure, was modulated by increasing concentrations of U-75302. Specifically, treatment with 0.5, 1.5 and 3.0 mg U-75302/100 g body wt reduced microvascular permeability by 17, 67 and 98%, respectively. In conclusion, LTB4 plays an important role in oedema formation in acute oxidative injury of the immature lung and this is mediated, in part, through neutrophils.

Animals↗
PubMed1994

In utero exposure to maternal low protein diets induces hypertension in weanling rats, independently of maternal blood pressure changes.

The association between maternal nutrition, fetal growth and the later development of hypertension was investigated in the rat. Animals were habituated to diets containing 18% (control) or 9% (low) protein by weight. The rats were mated and maintained on the diets until the end of pregnancy. Lactating dams were transferred onto standard chow diet. Systolic blood pressure was determined in male and female weanling offspring, using an indirect tail-cuff method. To assess the direct effects of low protein diets upon blood pressure of adult animals, a group of male and female rats were fed 18% or 9% protein for 14 days. Blood pressure was determined at the beginning and end of the feeding period. Blood pressure was additionally assessed over 14 days in pregnant rats fed control or low protein diets. Low protein diets did not alter systolic blood pressure in adult male or female rats. The blood pressures of pregnant females fed 18% or 9% protein diets did not significantly differ at any stage of pregnancy. Rats fed 9% protein diets gave birth to significantly smaller pups. Litter sizes were unaltered, and no differences in perinatal mortality were observed. Pups exposed to maternal low protein in utero had higher systolic blood pressure at the age of 4 weeks, when compared to control pups. The phenomenon was observed in both male and female offspring. Blood pressures at 4 weeks of age were strongly associated with maternal protein intake (r = -0.55). Associations were also noted between blood pressure and maternal weight at mating (r = 0.48), and weight gain in pregnancy (r = -0.30). Fetal exposure to maternal low protein diets induces hypertension in rats. The phenomenon is observed early in life and is independent of sex and the influence of maternal blood pressure. The low protein diet itself did not produce an increase in the blood pressure of adult rats.

Journal Article↗
PubMed1993

Dexamethasone treatment fails to reduce oxygen-induced lung injury in the preterm guinea pig. Effects on pulmonary inflammation and antioxidant status.

Dexamethasone (10 mg/kg/day) or vehicle was administered in a randomized, controlled fashion to 3-day preterm guinea pigs exposed to either 21% oxygen or 95% oxygen for 72 hr and maintained in room air for a further 96 hr. Treatment with dexamethasone had no effect on survival of preterm pups maintained in either 21% or 95% O2. Dexamethasone treatment reduced the growth rate of pups, the effect occurring earlier (0-3 days) in 21% O2-treated pups than in 95% O2-treated pups (5-7 days). Exposure to 95% O2 reduced the survival rate of preterm animals (73% vs 100%, P < 0.05). Surviving pups developed acute lung injury, characterized by the accumulation of a protein-rich exudate in the alveoli and an infiltration of inflammatory cells, particularly neutrophils into the lung. Dexamethasone treatment attenuated the pulmonary inflammatory cell infiltration, in particular neutrophils, both during oxygen exposure (16.4 x 10(4) vs 9.4 x 10(4)/mL; P < 0.05) and following return to ambient conditions (28.0 x 10(4) vs 5.1 x 10(4)/mL; P < 0.05). Elastase activity in bronchoalveolar lavage fluid, which was primarily of neutrophil origin, was unchanged by dexamethasone treatment. Dexamethasone-treated pups had increased pulmonary antioxidant enzyme activities (Cu/Zn-superoxide dismutase; Mn-superoxide dismutase, catalase and glutathione peroxidase) during recovery from oxidative injury. Although there was both a marked reduction in numbers of neutrophils in the lung and elevated pulmonary antioxidant enzyme activities in dexamethasone-treated pups, the degree of microvascular permeability, as determined by both the lung wet weight/dry weight ratio and the presence of plasma proteins in the lavage fluid, was unchanged. Combined, these results imply that dexamethasone, although capable of blunting the influx of neutrophils to the hyperoxia-exposed lung and inducing antioxidant defences in the immature lung, cannot modify the progression of acute oxygen-induced injury of the immature lung.

Amino Acid Sequence↗
PubMed1993

Temporal association between pulmonary inflammation and antioxidant induction following hyperoxic exposure of the preterm guinea pig.

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O2 (control) or 95% O2 (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.

Animals↗
PubMed1992

The E. coli ffh gene is necessary for viability and efficient protein export.

Homologues of the gene encoding the 54K (M(r) 54,000) subunit of the mammalian signal recognition particle have been identified in different organisms. The Escherichia coli homologue, termed ffh (for fifty-four homologue), specifies a protein (Ffh) that shares many properties with its eukaryotic counterpart, including association with mammalian 7S RNA and the ability to bind signal sequences specifically. Ffh also associates with E. coli 4.5S RNA, showing that it can form a ribonucleoprotein complex in prokaryotes. These results are intriguing because extensive genetic and biochemical characterization of E. coli failed to identify a signal recognition particle-like mechanism for protein export. Here we address this issue directly by construction of a strain in which ffh expression is arabinose-dependent. Results of depletion experiments indicate that Ffh is important in protein translocation.

Bacterial Proteins↗
PubMed1992

Dietary supplementation of vitamin E fails to prevent the development of hyperoxic lung injury in the premature guinea pig.

1. The benefit of dietary vitamin E supplementation in preventing oxidative-induced lung injury was investigated. Three day preterm guinea pig pups were exposed to hyperoxic (85% O2) or normoxic (21% O2) conditions. The animals were fed either a standard low birthweight human infant formula milk (6.4 mg/l vitamin E), or a vitamin E supplemented milk (100 mg/l) for up to 7 days. 2. After 3 days vitamin E supplementation, plasma but not erythrocyte vitamin E concentrations were elevated, while following 7 days both plasma and erythrocyte vitamin E concentrations were significantly increased. 3. Lung and liver vitamin E concentrations were elevated at both 3 and 7 days. At 3 days the increase in lung vitamin E was oxygen-dependent, suggesting that the lung increases uptake of vitamin E in response to oxidative stress. 4. Despite an increase in the vitamin E concentration of the lungs of preterm guinea pigs, no amelioration of the lung injury was observed. These results suggest that although vitamin E is a potent antioxidant, it is unable to protect adequately the lungs from reactive oxygen species in the absence of sufficient primary enzymatic antioxidant defences.

Animals↗
PubMed1992

Magnitude of hyperoxic stress and degree of lung maturity determine the nature of pulmonary antioxidant response in the guinea pig.

The ability of the immature lung to induce antioxidant defences in response to hyperoxic stress was examined. Preterm guinea pigs (65 days gestation, term = 68 d) were exposed to either 21% O2, 85% O2 or 95% O2 for 72 hours. Exposure to 85% O2 increased lung catalase, glutathione peroxidase and manganese superoxide dismutase activities in comparison to air controls. Exposure to 95% O2 resulted only in an increase in glutathione peroxidase activity. Bronchoalveolar lavage fluid GSH concentration was increased by a similar amount by both exposure regimes, while lung copper/zinc superoxide dismutase activity was unchanged by either treatment. Comparison of the antioxidant response of term and preterm animals exposed to 85% O2 for 72 hours indicated a greater response in the lung of the preterm animals. Manganese superoxide dismutase activity was elevated in both term and preterm animals, while catalase and glutathione peroxidase activities were elevated only in preterm animals. The extent of microvascular permeability as indicated by bronchoalveolar lavage fluid protein concentration, was lower in preterm animals than in term animals. We conclude that the immature lung can respond to hyperoxic stress by antioxidant induction and that the nature of the response is dependent, in part, both on the severity of the stress and on the maturity of the lung.

Animals↗
PubMed1991

Urinary desmosine, elastolysis, and lung disease.

Desmosine is an amino acid specific to elastin. Animal studies suggest that urinary desmosine (UD) represents endogenous elastin degradation. Therefore, UD has previously been used to investigate endogenous elastolysis, but was not elevated in subjects with chronic obstructive airways disease (COAD), although accelerated pulmonary elastolysis is thought to contribute to COAD. We have investigated whether this reflects large day-to-day and between-subject variation in UD and whether, in man, dietary desmosine contributes significantly to that in urine. Mean 24-hour UD output (over 5 consecutive days) from 10 asymptomatic subjects (5 males) was higher in males than females (77.4 +/- 9.6 and 40.2 +/- 5.0 nmol/24 hours, respectively; mean +/- SD, P less than .001), but not significantly different when expressed in terms of creatinine (micrograms desmosine/100 mg creatinine: males, 2.5 +/- 0.4; females, 3.1 +/- 0.8; mean +/- SD). The lowest between-subject variation was observed when the mean of 5 days' 24-hour UD values was analyzed on the basis of gender (coefficient of variation [CV], 12.5%); when gender was not considered, the least between-subject variation was found for the mean of 5 days' desmosine/creatinine analysis (CV, 24.5%). Approximately 1% of dietary desmosine (ingested as [3H]elastin and [3H] desmosine) was excreted in the urine within 24 hours, contributing approximately 15% of UD while on a normal diet. Although ingestion of a low elastin diet (less than 1/10 desmosine/24 hours than a normal diet) resulted in lower within-subject variation in 24-hour UD excretion (mean CV decreased from 31.5% to 20.2%), the between-subject CV and UD levels did not alter.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

The pre-term guinea-pig: a model for the study of neonatal lung disease.

1. Research into the pathogenesis of acute and chronic neonatal lung disease has been hampered by the lack of a suitable small-animal model of prematurity. We describe such a model that has been developed and validated in the guinea-pig. 2. Pre-term guinea-pigs delivered by Caesarian section at 65 days gestation (normal gestation 68 days) exhibited transient respiratory distress. The survival of pre-term animals was lower than that of term animals after exposure to 95% O2 (pre-term 42% versus term 79% at 96 h, P less than 0.05). 3. Pulmonary histology in pre-term animals exposed to both 21% O2 and 95% O2 revealed evidence of acute lung injury with atelectasis, pulmonary oedema, fibrin deposition and inflammatory cell infiltration. No evidence of lung injury was observed in term animals exposed to 21% O2, whereas those exposed to 95% O2 showed a similar, but less pronounced, injury to that seen in pre-term pups. 4. The protein concentration in bronchoalveolar lavage fluid was similar in pre-term and term animals exposed to 95% O2, but neutrophil numbers in bronchoalveolar lavage fluid tended to be greater in pre-term pups. 5. Elastase-like activity, measured against succinyl-1-trialanine p-nitroanilide, was higher in bronchoalveolar lavage fluid from control pre-term animals compared with that from control term animals. Exposure to 95% O2 increased the elastase-like activity significantly in both groups. The majority of the elastase-like activity was EDTA-sensitive and thus is possibly due to metallo-elastase. Fractionation of bronchoalveolar lavage fluid indicated that the elastase-like activity was associated with a high-molecular-mass complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli.

The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms. One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ. In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E. coli, conferring a Lac+ phenotype. beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope. We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.

ATP-Binding Cassette Transporters↗
PubMed1990

The sec and prl genes of Escherichia coli.

Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec, secretion). The other identifies dominant suppressors of signal sequence mutations (prl, protein localization). Subsequent characterization reveals that in at least three cases, prlA/secY, prlD/secA, and prlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.

Bacterial Proteins↗
PubMed1990

Increased expression of the bifunctional protein PrlF suppresses overproduction lethality associated with exported beta-galactosidase hybrid proteins in Escherichia coli.

We have cloned and determined the nucleotide sequence of the prlF gene. An open reading frame predicting a 111-amino-acid protein (Mr 12,351) with an acidic carboxy terminus was identified. The DNA sequence preceding this open reading frame revealed a putative promoter and a ribosome-binding site. The nucleotide sequence of the prlF1 mutation revealed a 7-base-pair duplication resulting in a slightly smaller predicted gene product of Mr 12,009 that lacked the acidic carboxy terminus. Maxicell analysis of prlF and prlF1 subclones identified peptides of sizes similar to those predicted by the nucleotide sequences. The prlF sequence was shown to be expressed in vivo by both maxicell analysis and construction of a prlF-lacZ fusion. Two kanamycin resistance insertions within the prlF open reading frame were introduced into the chromosome, replacing the wild-type gene. In contrast to the prlF1 mutation, these insertions had no detectable effect on cell growth or on the beta-galactosidase activity or maltose sensitivity (two sensitive indicators of hybrid protein export) conferred by the lamB-lacZ42-1 gene fusion. Overproduction of the wild-type prlF gene product from a plasmid carrying an active hybrid promoter, however, conferred a prlF1 phenotype. In addition, both the prlF1 mutation and both kanamycin resistance insertions increased the beta-galactosidase activity of a prlF-lacZ fusion. These results suggest that prlF is autoregulated and that overproduction of the prlF gene product increases the export efficiency of beta-galactosidase hybrid proteins from the cytoplasm.

Bacterial Proteins↗
PubMed1988

Emphysema in the Blotchy mouse: a morphometric study.

Inheritance of the Blotchy allele at the X-chromosomal Mottled locus of mice results in changes in the lung which resemble emphysema. Previous studies using measurements of mean linear intercept have recognized emphysema in the hemizygous, Blo/Y, male and homozygous, Blo/Blo, female, but not in the heterozygous, Blo/+, female. The aim of this study was to develop a rapid, accurate method for analysis of emphysema and to establish whether it would identify emphysema in the heterozygote. Lungs from wild type and outbred Blo mice were inflated with fixative at a pressure of 25 cm and then sectioned and stained. Transect lengths across air spaces were measured using computerized image analysis and the results were plotted as histograms. Data were also expressed as cumulative frequencies (ogives) and subjected to statistical analysis by the non-parametric Kolmogorov-Smirnov test. Emphysema was shown by significantly increased (P less than 0.001) transect lengths in the Blo/Blo female and Blo/Y male compared with the wild type controls. The heterozygous Blo/+ mice form an intermediate group significantly different (P less than 0.001) from the wild and blotchy mice. The sensitivity of the method is illustrated by the fact that the Blo/+ female formed an intermediate group between wild type and the Blo/Y male and Blo/Blo female.

Animals↗
PubMed1988

Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12.

In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction. This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V). We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products. Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA. The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene. Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene. A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains. Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts. The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.

Alleles↗