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Biomedical subjects

H J Barnes

Publications and source records attributed to H J Barnes.

At least 19 recordsLinked to original sources
PubMed2000

Poult enteritis complex.

Poult enteritis complex (PEC) is a general term that encompasses the infectious intestinal diseases of young turkeys. Some diseases, such as coronaviral enteritis and stunting syndrome, are relatively well characterised, while others, such as transmissible viral enteritis, poult growth depression and poult enteritis mortality syndrome, remain ill-defined. All forms of PEC are multifactorial, transmissible and infectious. Salient clinical features include stunting and poor feed utilisation that result from enteritis. In the more severe forms, runting, immune dysfunction and mortality are reported. Gross and microscopic lesions of enteritis are present in all forms but tend to be non-specific. Other lesions may be present, depending on the agents involved. The basic pathogenesis involves the following: a) alteration of the intestinal mucosa, generally by one or more viruses infecting enterocytes; b) inflammation; c) proliferation of secondary agents, usually bacteria. Non-infectious factors interplay with infectious agents to modulate the course and severity of disease. Diarrhoea is believed to be primarily osmotic because of maldigestion and malabsorption, but may also have a secretory component. Transmission is primarily faecal-oral. No public health significance is recognised or suspected. Prevention is based on eliminating the infectious agents from contaminated premises and preventing introduction into flocks. This is accomplished by an effective cleaning, disinfection and biosecurity programme. All-in/all-out production or separate brooding and finishing units are helpful. Control may require regional co-ordination among all companies producing turkeys, especially if the production is highly concentrated, and a quarantine programme for more severe forms of PEC. No vaccines or specific measures for controlling the organisms involved in PEC are available. Treatment is supportive for the viral component, while antibiotics, especially those with efficacy against Gram positive bacteria, may help to reduce the impact to bacterial infections. Evidence suggests that PEC occurs wherever turkeys are raised commercially, but this is not well documented and distribution of the various organisms that have been associated with PEC is largely unknown. The disease causes enormous economic loss, mostly from failure of the turkey to reach its genetic potential.

Animals↗
PubMed1998

Regulation of CCSP (PCB-BP/uteroglobin) expression in primary cultures of lung cells: involvement of C/EBP.

The Clara-cell secretory protein (CCSP) is a cell-specific differentiation marker for the bronchiolar Clara cell. Isolated rat Clara and alveolar type 2 cells kept in primary culture proliferate and dedifferentiate, providing the opportunity to study differentiation-dependent mechanisms. In freshly isolated Clara cells, high levels of CCSP and the corresponding mRNA were detected. During culture in vitro, these levels decreased. In the type 2 cell fraction, low levels of CCSP were detected, which decreased further during culture. A promoter fragment of the rat CCSP gene encompassing the sequence from -188 to +53 was able to drive high-level expression of reporter genes in transfected Clara cells. Reporter gene expression in transfected type 2 cells was markedly lower, and no expression could be detected in alveolar macrophages. Expression of transcription factors previously described to stimulate CCSP expression appeared not to parallel CCSP levels in the primary Clara cells. However, expression of the transcription factor C/EBP alpha correlated with the CCSP expression pattern. In electrophoretic mobility shift assays, we were able to demonstrate binding of C/EBP alpha from rat Clara cell nuclear extracts to an element located 85 bp upstream of the start site of transcription. Overexpression of C/EBP alpha increased expression from the CCSP -188 promoter fragment up to fivefold in NCI-H441-cells and 30-fold in A549-cells, establishing the functional importance of C/EBP alpha. Our results show that primary cultures of Clara cells constitute a useful model for investigating terminal airway differentiation and suggest a role for C/EBP-factor(s) in this process.

Animals↗
PubMed1998

Intervention decision model to prevent spiking mortality of turkeys.

Based on the daily records on turkeys' mortalities for the series of flocks placed on different farms in a relatively compact geographical area for the period of approximately 2 yr and other relevant explanatory variables, the goal of the research was to design a decision model to determine whether or not to use the fluorquinolone antibiotic, sarafloxacin, to prevent spiking mortality of turkeys. The core of the designed decision model is the forecasting model that attempts to ex-ante predict the cumulative flock mortality for the period between 8 and 28 d of age. Forecasts were generated with the parameters of the linear regression model where continuous values of daily mortalities served as a dependent variable. The decision variable is a binary (yes/no) choice variable, where "yes" means "go ahead with treatment" and "no" means "do nothing". If the predicted cumulative mortality for the period between 8 and 28 d of age exceeds 9% of the total initial placement, the model generates a "yes" signal. If the predicted cumulative mortality for the same period is below 9% of the total initial placement, the model generates a "no" signal. The results indicate a reasonable accuracy of the prediction model where the number of correct prediction increases and the number of incorrect predictions falls very fast as the forecasting window shortens. The intervention decision model could help veterinarians in making decisions on whether or not to treat the suspect flocks.

Aging↗
PubMed1998

Bordetella avium virulence measured in vivo and in vitro.

Bordetella avium causes an upper-respiratory-tract disease called bordetellosis in birds. Bordetellosis shares many of the clinical and histopathological features of disease caused in mammals by Bordetella pertussis and Bordetella bronchiseptica. In this study we determined several parameters of infection in the domestic turkey, Meleagris galapavo, and compared these in vivo findings with an in vitro measure of adherence using turkey tracheal rings. In the in vivo experiments, we determined the effects of age, group size, infection duration, and interindividual spread of B. avium. Also, the effect of host genetic background on susceptibility was tested in the five major commercial turkey lines by infecting each with the parental B. avium strain and three B. avium insertion mutants. The mutant strains lacked either motility, the ability to agglutinate guinea pig erythrocytes, or the ability to produce dermonecrotic toxin. The susceptibilities of 1-day-old and 1-week-old turkeys to B. avium were the same, and challenge group size (5, 8, or 10 birds) had no effect upon the 50% infectious dose. Two weeks between inoculation and tracheal culture was optimal, since an avirulent mutant (unable to produce dermonecrotic toxin) persisted for a shorter time. Communicability of the B. avium parental strain between confined birds was modest, but a nonmotile mutant was less able to spread between birds. There were no host-associated differences in susceptibility to the parental strain and the three B. avium mutant strains just mentioned: in all turkey lines tested, the dermonecrotic toxin- and hemagglutination-negative mutants were avirulent whereas the nonmotile mutants showed no loss of virulence. Interestingly, the ability of a strain to cause disease in vivo correlated completely with its ability to adhere to ciliated tracheal cells in vitro.

Animals↗
PubMed1997

Disruption of short-duration timing associated with damage to the suprachiasmatic region of the hypothalamus.

The neural bases of circadian rhythmicity have been demonstrated in a variety of animal species, including primates. Yet, the brain mechanisms underlying time experience and the timing of behaviors of shorter duration are still not well understood. In the present study, we demonstrate disruption of short-duration timing capacity in AH, a patient with damage to the suprachiasmatic (SCN) region of the hypothalamus. AH exhibited extreme inconsistency in her rate of tapping production on a motor continuation paradigm. Her inter-response intervals (IRIs) were extremely large compared with normal control subjects and were similar to those previously reported in patients with cerebellar dysfunction. Increased variability of both central timing and motor implementation processes was evident compared with both age-matched and elderly normal control subjects. Severe impairment of time perception was also evident on duration discrimination, whereas auditory loudness discrimination was intact. These findings suggest that a hierarchic relationship between long-duration (circadian) and short-duration timing exists, and that in addition to the cerebellum, intact hypothalamic functioning is necessary for short-duration timing.

Adult↗
PubMed1996

Structural basis for calcium binding by uteroglobins.

Uteroglobins, i.e. proteins with similar three-dimensional structure and ligand binding specificity to uteroglobin from rabbit uterus, have been found in rat, mouse and human lung. We have recently demonstrated the binding of calcium by human uteroglobin, and we have therefore tried to find potential binding sites for metals in the three-dimensional structure of uteroglobin by the use of two different computational procedures. A putative binding site for calcium in uteroglobin was identified by means of a hydrophobic contrast function. The spatial disposition of atoms that could ligand calcium in the putative calcium-binding site appears similar to that of the primary calcium-binding site of secretory phospholipase A2 enzymes, consisting of the carboxyl group of an aspartic acid residue and a loop providing three backbone carbonyl oxygens. From inspection of their primary sequences and three-dimensional structures, it became clear that this putative calcium-binding motif is conserved among uteroglobins from different species. The potential significance of the predicted site was investigated by site-directed point mutagenesis of human uteroglobin in which Asp46 was replaced by Asn or Lys. In both mutants, the ruthenium red and 45Ca2+ binding was significantly reduced. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis under non-reducing conditions indicated that the mutant proteins had the expected molecular masses and that their ability to dimerize was not disturbed by these mutations. Valence calculations also identified the putative calcium-binding site, but only after optimization of its conformation by the use of molecular dynamics with a restrained calcium ion. Our results support the notion that Asp46 of uteroglobins acts as a "cap" residue in a calcium-binding site structurally similar to the primary calcium binding sites of phospholipases A2.

Amino Acid Sequence↗
PubMed1995

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Animals↗
PubMed1995

Solution structure of a mammalian PCB-binding protein in complex with a PCB.

Metabolites of polychlorinated biphenyls (PCBs) bind with high affinity to uteroglobin, a small homodimeric protein that also binds progesterone. We present the solution structure of the reduced form of rat uteroglobin in complex with a PCB methylsulphone, (MeSO2)2-TCB. The structure reveals the molecular basis for the accumulation of (MeSO2)2-TCB by uteroglobin. The structure also shows how ligand binding and release might be controlled by reduction/oxidation of two intermolecular disulphide bonds. Breakage of these bonds induces a local unfolding of the N- and C-termini and a separation of helices creating a channel into the binding site. These effects make the ligand binding cavity readily accessible to entry of the ligand.

Amino Acid Sequence↗
PubMed1994

Heterologous expression of human uteroglobin/polychlorinated biphenyl-binding protein. Determination of ligand binding parameters and mechanism of phospholipase A2 inhibition in vitro.

High level expression of a human polychlorinated biphenyl-binding protein (hPCB-BP; also termed uteroglobin or CC10) was achieved in Escherichia coli. The recombinant protein (rhPCB-BP) constituted approximately 1% of total bacterial lysate proteins as judged from in vitro ligand binding assays using 4,4'-bis([3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl. rhPCB-BP was purified to homogeneity in its native dimeric form. Saturation analysis experiments indicated a Kd of approximately 69 nM for the binding of 4,4'-bis([3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl to rhPCB-BP. The average number of binding sites (Bmax) calculated from such experiments on purified rhPCB-BP was 49 nmol/mg of protein and is close to the theoretical value of 1 mol of ligand associating with 1 mol of dimeric protein. Purified rhPCB-BP was also found to cause a dose-dependent inhibition of the enzyme porcine pancreatic phospholipase A2 (PLA2) in vitro. Increasing the concentrations of calcium abolished the inhibition of PLA2 by rhPCB-BP, suggesting that the protein functions in vitro by sequestering Ca2+, an essential PLA2 cofactor. This notion was further supported by direct evidence that 45Ca2+ binds to rhPCB-BP. 1 mol of dimeric protein was also found to bind 2 mol of ruthenium red, an organic dye that detects Ca(2+)-binding proteins, with a Kd of 3 microM. This binding was inhibited by Ca2+, with an IC50 of 7 mM. Finally, it was demonstrated that the addition of a high affinity ligand for the protein had no effect on its ability to inhibit PLA2 under conditions of limiting concentrations of calcium, and the addition of Ca2+ did not affect the binding characteristics of the PCB ligand, suggesting that these two properties of the protein are independent. Our results strongly support the notion that ligand binding is a conserved feature of the homologous uteroglobin/PCB-BP/cc10 proteins in different species, whereas our results question the suggested role of these proteins as specific inhibitors of PLA2.

Calcium↗
PubMed1994

Baculovirus expression of bovine cytochrome P450c17 in Sf9 cells and comparison with expression in yeast, mammalian cells, and E. coli.

Expression of bovine 17 alpha-hydroxylase cytochrome P450 (P450c17) in insect Sf9 cells using baculovirus is the fourth heterologous expression system developed for studying this enzyme. The enzyme in Sf9 cell membranes has all the expected activities and closely resembles that from the other systems: COS1 cells, yeast, and Escherichia coli. One curious feature of baculovirus expression of this hemoprotein is that the immunodetectable P450c17 is present in an insoluble pellet when produced in the absence of added hemin and in a 10,000 g supernatant when expressed in the presence of added hemin. When comparing the level of expression of bovine P450c17 in these different expression systems, E. coli produces the largest quantity per liter culture, while baculovirus produces the most molecules of P450c17 per cell.

Animals↗
PubMed1993

Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.

Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.

Amino Acid Sequence↗
PubMed1993

Expression in Escherichia coli of functional cytochrome P450c17 lacking its hydrophobic amino-terminal signal anchor.

A truncated form of bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P450c17) lacking its amino-terminal hydrophobic signal anchor sequence (delta 2-17) was expressed in Escherichia coli to give more than 600 nmol P450 per liter using XL1-blue as the host. The expression level of the truncated P450c17 showed variation among different host strains. When intact E. coli cells were used for analysis, the truncated P450c17 showed the typical cytochrome P450 CO-difference spectrum and type I substrate binding spectra for pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, and 17 alpha-hydroxyprogesterone. The apparent Ks values for these steroids are comparable to those obtained under the same conditions with wild type P450c17. The truncated P450c17 is primarily associated with the membrane fraction from E. coli and remained with this fraction after treatment with 0.1 M Na2CO3. The 17 alpha-hydroxylase activity of the truncated form was observed using intact E. coli. Using isolated membrane fractions, the truncated P450c17 also showed 17,20-lyase activity upon reconstitution with rat liver microsomal NADPH-cytochrome P450 reductase. These results indicate that P450c17 can be associated with E. coli membranes not only via the amino-terminal hydrophobic region as expected for the wild type, nontruncated form, but also through other sequences within the polypeptide chain and that integration of the amino-terminal region into the membrane is not essential for the folding pathway leading to functional P450c17 in E. coli. This is in contrast to mammalian cells where the same truncated P450c17 is found to be inactive and the folding pathway leading to functional P450c17 appears to require a signal anchor sequence.

Animals↗
PubMed1992

Time course of movement planning: selection of handgrips for object manipulation.

A goal of research on the cognitive control of movement is to determine how movements are chosen when many movements are possible. We addressed this issue by studying how subjects reached for a bar to be moved as quickly as possible from a home location to a target location. Ss generally grabbed the bar in a way that afforded a comfortable posture at the target location (the end-state comfort effect) and with the thumb toward the end of the bar that would be aligned with the target (the thumb-toward bias). The data suggested that subjects chose handgrips by retrieving instances of previous reaches, not by carrying out computations that treated candidate reaches as new behavioral events.

Acceleration↗
PubMed1992

Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate.

Ninety commercial broiler chickens were divided into three equal groups; 30 were injected with brain-heart-infusion broth into the cranial thoracic air sacs (controls), 30 were similarly inoculated with a culture of Escherichia coli, and 30 were similarly inoculated with E. coli cell-free culture filtrate. Birds were examined from 0 to 6 hours post-inoculation. E. coli-inoculated and cell-free culture filtrate-inoculated chickens reacted similarly, with exudation of heterophils into the air sac. Microscopically, heterophils were present in low numbers perivascularly 0.5 hour after inoculation and became more numerous by 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, air sac epithelial cells were swollen and vacuolated, and interdigitating processes were separated. Histologically and ultrastructurally, all features in control chickens were normal, with only rare heterophils in the air sac interstitium. In E. coli-inoculated and cell-free culture filtrate-inoculated chickens, cell counts (predominantly heterophils) in air sac lavage fluids increased markedly at 3 and 6 hours, with only slight increases in counts from lavages of controls. Heteropenia was observed in E. coli-inoculated chickens, whereas heterophilia was observed in cell-free filtrate chickens and controls. Ninety additional chickens were pretreated with cyclophosphamide, subdivided into three equal groups, and inoculated and examined similarly as above. Cyclophosphamide pretreatment reduced inflammatory changes in air sacs, lowered cell numbers in lavage fluids, and abolished hematologic changes; however, it did not prevent epithelial cell changes. These results indicate that cell-free culture filtrate of E. coli induces changes similar to those induced by cultures of E. coli.

Acute Disease↗
PubMed1992

Superior vena cava syndrome: etiology, diagnosis, and treatment.

Superior vena cava (SVC) syndrome is a critical condition in which an intrathoracic mass lesion compresses the SVC and promotes the development of head and upper body edema and cyanosis. SVC syndrome develops in 10% of patients with a right-sided malignant intrathoracic mass lesion. Diagnostic evaluation and emergency therapy are always necessary to assess and alleviate airway obstruction, cerebral venous hypertension and symptoms secondary to mediastinal compression. Radiation therapy and venous bypass of the obstructed SVC are both used successfully as early treatment. Although radiation therapy to the malignant process may provide initial decompression, a more sustained decrease in venous pressure occurs in patients who also undergo decompressive SVC surgical bypass. SVC bypass should be considered early in the course of patients with profound cerebral or laryngeal edema, patients with extensive thrombosis of the SVC, and in rare patients afflicted with severe venous hypertension and in whom a tissue diagnosis requires a mediastinal exploration.

Adrenal Cortex Hormones↗
PubMed1991

Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P450scc) in Escherichia coli.

Escherichia coli expression vectors containing the trc promoter and the complete DNA sequence of either the precursor or the mature form of bovine adrenocortical cholesterol side chain cleavage cytochrome P450 (P450scc) were transformed into E. coli strain JM109 and transcription induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Immunoreactive cytochrome P450scc was produced using the plasmid containing the mature P450scc sequence but not with the plasmid containing the sequence of the precursor form of P450scc, even though P450scc RNA was detectable in both cases. The mature form of P450scc was detected spectrophotometrically in a reduced CO-difference spectrum in E. coli (40-60 nmol/liter culture). Cholesterol and hydroxylated derivatives (22-hydroxycholesterol and 25-hydroxycholesterol) produce a type 1 substrate-binding spectrum in IPTG-induced, transformed E. coli. The P450scc was found to be associated with the E. coli membrane fraction and the enzymatic activity of side chain cleavage of 25-hydroxycholesterol was reconstituted using solubilized membranes, in the presence of purified bovine adrenocortical adrenodoxin and NADPH-adrenodoxin reductase (turnover number; 15.4 nmol/min/nmol P450). This bacterial expression system provides functional P450scc, in the absence of other forms of P450, which can be used for evaluation of enzymatic and spectral properties of this mitochondrial P450 by site-directed mutagenesis.

Animals↗
PubMed1991

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

17-alpha-Hydroxypregnenolone↗