Molecular and cellular targeting in the expression of foreign polypeptides in bacteria.
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ATP-Binding Cassette Transporters↗
Biomedical subjects
D O'Callaghan
Publications and source records attributed to D O'Callaghan.
The effects of verapamil on training in patients with ischemic heart disease.
Verapamil is a calcium-channel blocking agent with antianginal and antiarrhythmic properties that have been widely studied. Its myocardial depressant effect is well known. The purpose of this study was to examine the effects of verapamil on the training response in patients with ischemic heart disease. The study group consisted of 41 male patients with a mean age of 53.3 +/- 7.2 years who had suffered a myocardial infarction or had undergone coronary artery bypass surgery 8 to 12 weeks previously. They were chosen on a consecutive basis from eligible patients entering a cardiac rehabilitation program. With use of a double-blind technique, 21 patients were assigned to receive verapamil, 120 mg three times daily, while the other 20 were given an identical placebo. Each patient underwent exercise stress testing in the untreated state to permit comparison between tests performed on commencement and completion of training. The training effect was determined by comparing exercise response before and after the eight-week program. There was an increase in exercise duration (p less than 0.001) and a decrease in functional aerobic impairment (p less than 0.001), without difference between the two groups. Energy expenditure increased in both groups, but the highest level was achieved by those receiving active treatment (p less than 0.02). Heart rate for equal workload was significantly reduced after training (p less than 0.001), although this was lower in the placebo patients (p less than 0.001) and the patients who had a recent myocardial infarction (p less than 0.01). It appears that treatment with verapamil does not impair the development of a training effect in patients with ischemic heart disease who are undergoing organized training.
Anaerobic Threshold↗
Variation in the timing of the reproductive season among breeds of sheep in relation to differences in photoperiodic synchronization of an endogenous rhythm.
Photoperiod may regulate seasonal reproduction either by providing the primary driving force for the reproductive transitions or by synchronizing an endogenous reproductive rhythm. This study evaluated whether breed differences in timing of the reproductive seasons of Finnish Landrace (Finn) and Galway ewes are due to differences in photoperiodic drive of the reproductive transitions or to differences in photoperiodic synchronization of the endogenous rhythm of reproductive activity. The importance of decreasing photoperiod after the summer solstice in determining the onset and duration of the breeding season was tested by housing ewes from the summer solstice in either a simulated natural photoperiod or a fixed summer-solstice photoperiod (18 h light:6 h dark; summer-solstice hold). Onset of the breeding season within each breed did not differ between these photoperiodic treatments, but Galway ewes began and ended their breeding season earlier than Finn ewes. The duration of the breeding season was shorter in Galway ewes on summer-solstice hold than on simulated natural photoperiod; duration did not differ between photoperiodic treatments in Finn ewes. The requirement for increasing photoperiod after the winter solstice for initiation of anoestrus was tested by exposing ewes from the winter solstice to either a simulated natural photoperiod or a winter-solstice hold photoperiod (8.5 h light:15.5 h dark). Onset of anoestrus within each breed did not differ between these photoperiodic treatments, but the time of this transition differed between breeds. These observations suggest that genetic differences in timing of the breeding season in Galway and Finn ewes do not reflect differences in the extent to which photoperiod drives the reproductive transitions, because neither breed requires shortening days to enter the breeding season or lengthening days to end it at appropriate times. These findings are consistent with the hypothesis that photoperiod synchronizes an endogenous rhythm of reproductive activity in both breeds and that genetic differences in timing of the breeding season reflect differences in photoperiodic synchronization of this rhythm.
Anestrus↗
Metabolic changes during serial squash matches in older men.
We have previously reported dramatic changes in heart rate and blood biochemistry in older men during and shortly after competitive squash. In this study we sought to determine whether these changes are attenuated or exaggerated during tournament matches played in rapid succession. Ten veteran (greater than 45 yrs) players were studied during three competitive matches played over a 36-hr period. Squash was associated with significant changes in heart rate and circulating concentrations of catecholamines, lactate, free fatty acids, and potassium. These changes were of equal magnitude and in some cases tended to be exaggerated during the second and third matches. These data confirm the acute changes in cardiac function and metabolism that occur during competitive squash and suggest that these responses are not down-regulated but may in fact be accentuated during sequential tournament matches.
Aging↗
Medical management of acute myocardial infarction in Ireland: information from the Second International Study of Infarct Survival (ISIS-2).
We report the management of the 831 patients from 18 hospitals which constituted the Irish component of the 17,183 subjects enrolled in the ISIS-2 trial which studied the effects of streptokinase infusion and aspirin therapy given to patients presenting within 24 hours of the onset of suspected acute myocardial infarction. 34% of Irish patients (IP) presented for treatment within 4 hours of the development of symptoms. This compared to 44% of the overall group (OG) (p less than 0.001). This represented the lowest percentage of patients presenting within 4 hours in any of the participating countries. The mean delay time from onset of symptoms was 7.9 hours in IP compared to 6.9 in OG (p less than 0.001). The mean delay time in Ireland was longer than the mean delay time in any of the participating countries. The mean age and systolic blood pressure at presentation was similar in both groups. It was planned to treat 12% IP with aspirin compared to 10% OG (p-NS), and 71% IP with subcutaneous heparin compared with 47% OG (p less than 0.001). Intravenous heparin was planned treatment in 20% IP and 24% OG (p less than 0.01). Planned oral anticoagulant therapy was similar in both groups (p-NS). Planned use of intravenous betablockers occurred in only 2% IP and 6% OG (p less than 0.001). 9% IP and 22% OG received steroids before streptokinase infusion (p less than 0.001). 65% IP and 47% OG received subcutaneous heparin (p less than 0.001). The use of intravenous heparin and oral anticoagulants was similar in IP and OG (p-NS).(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic beta-Antagonists↗
A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis.
We present a simple and flexible procedure to elicit and assay anti-peptide antibodies without peptide synthesis. It consists of expressing the peptide of interest in the form of a genetic insert within two different "recipient" bacterial proteins. One hybrid protein is used as immunogen for the induction of antibodies against the inserted peptide and the other as antigen for monitoring the anti-peptide antibodies raised. The two "recipient" proteins used are the MalE and the LamB proteins from E. coli. The MalE hybrid proteins can be affinity purified on an amylose column using mild nondenaturing conditions and can be crystalized for structural studies; LamB hybrid proteins express the inserted peptide on the cell surface so that intact bacteria can be used as a reagent. We chose, as a model peptide, a B-cell epitope from the pre-S(2) region of Hepatitis B virus. With both MalE and LamB hybrid proteins, high titres of anti-preS antibodies, able to react with native HBsAg particles, were induced in mice. The anti-peptide antibody titres recorded by ELISA were comparable to those obtained when either a synthetic peptide, or the hybrid proteins, were used as immobilized antigen.
Animals↗
Is there a neutralization epitope in the second conserved domain of HIV-1 envelope protein?
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Amino Acid Sequence↗
Role of short days in timing the onset and duration of reproductive activity in ewes under artificial photoperiods.
The objective of this work was to evaluate the role of short photoperiod in timing the onset and duration of reproductive activity in ewes. The perception of photoperiod was disrupted by pinealectomy following transfer from long (17L:7D) to short (8.5L:15.5D) photoperiod and the subsequent reproductive response was monitored. Ovariectomized ewes given Silastic implants containing estradiol-17 beta were exposed to long days until Day 0 (May 24) and then were allocated to the following groups (n = 5-6/group): Group 1) short-day control--moved to short days; Groups 2 to 5) pinealectomy after 0, 30, 60, or 90 short days, respectively; Group 6) long-day hold--kept on long days; Group 7) long days after 60 short days--moved to short days on Day 0 and returned to long days on Day 60. Six ewes kept outdoors served as additional controls. Reproductive neuroendocrine activity was assessed from plasma LH concentrations, high values being indicative of the breeding season and low values indicative of anestrus. Time of reproductive neuroendocrine activity onset (LH rise) did not differ among animals in the 7 groups kept indoors, but was advanced (p less than 0.05) relative to that of ewes outdoors. In contrast, duration of the LH elevation differed among ewes in groups kept indoors.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
What photoperiodic signal is provided by a continuous-release melatonin implant?
The purpose of this study was to evaluate whether the insertion of a continuous-release melatonin implant into ewes provides a short-day photoperiodic signal or acts as a functional pinealectomy (provides no specific photoperiodic signal but renders ewes incapable of responding to changes in photoperiod). Ewes primed with 60 long days (18L:6D) during the spring were moved to intermediate day length (13L:11D) for 66 days and then given one of five treatments: 1) short-day control, second drop in photoperiod to 8L:16D; 2) intermediate-photoperiod control, kept on 13L:11D; 3) pinealectomy and kept on 13L:11D; 4) melatonin implant and kept on 13L:11D; 5) melatonin implant and moved to 8L:16D. Mean number of estrous cycles per group and total duration of reproductive activity were determined. Ewes in all groups began to exhibit estrous cycles after the initial reduction in photoperiod. The number of estrous cycles and duration of reproductive activity differed among groups. The number of estrous cycles and duration of reproductive activity was extended in ewes receiving the second drop in photoperiod compared to that of the intermediate-photoperiod controls. Pinealectomized ewes had a number of estrous cycles and duration of reproductive activity similar to those of ewes maintained on the intermediate photoperiod. Melatonin implants increased the number of estrous cycles and prolonged reproductive activity in ewes maintained on the intermediate photoperiod; melatonin implants did not prevent the extension of reproductive activity in ewes receiving the second photoperiodic drop to the short daylength.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effect of bovine somatotropin on production and reproduction in prepubertal Friesian heifers.
This experiment was conducted to evaluate the effect of bST on average daily gain, onset of puberty, first lactation milk yield, and reproductive efficiency in Friesian heifers. Heifers (n = 16 per treatment) were allocated to either: 1) control (1.5 ml of vehicle) or 2) bST (15 mg of bST in 1.5 ml of vehicle) using a randomized complete block design. Subcutaneous injections of bST were administered once daily from 7 mo of age for 120 d or until heifers reached puberty. Heifers were weighed every 2 wk, and blood samples were collected twice weekly after heifers reached 200 kg. Progesterone concentrations were used to determine onset of puberty. Heifers were bred between 16 and 18 mo of age and, following parturition, milk yield and composition were recorded twice weekly and once every 2 wk, respectively. Heifers assigned to bST treatment had an average daily gain (kg) of .8 compared with .7 in control heifers. Number of days from birth to onset of puberty for bST-treated heifers was 401 compared with 381 for control heifers. Treatment with bST had no effect on milk yield, milk composition, or reproductive efficiency during the first lactation. These data demonstrate that daily administration of bST to Friesian heifers from 7 to 11 mo of age does not affect average daily gain, onset of puberty, reproductive efficiency, or first lactation milk yield of heifers.
Animals↗
Bacterial vectors to target and/or purify polypeptides: their use in immunological studies.
The construction of recombinant proteins by genetic engineering has opened new avenues in basic research (studies on protein organization, protein folding, immunogenicity of proteins, ...) and many different applications. Recombinant proteins which keep properties of both parental proteins are especially interesting. For example, if one protein--the vector protein--is targeted to a given cellular compartment, the other protein--the passenger--may be identically targeted. Also, if the vector protein can be purified by a simple affinity chromatographic procedure, this property may be extended to the passenger. The authors have developed a genetic procedure to detect "permissive" sites within potential vector proteins so that genetic fusion to these sites keep most or all biological properties of the vector. When they used LamB, an outer membrane protein from E. coli, foreign sequences could be expressed at the bacterial cell surface. This may lead to several types of applications: live bacterial vaccines, simple diagnostic tests, selection procedures for peptides with biological activity. When they used the MalE protein, a periplasmic maltose binding protein from E. coli, the passengers could be exported and purified in one-step high affinity chromatography in mild non-denaturing conditions. This led us to a simple preparation and purification scheme for the soluble part of the CD4 receptor for the Human Immunodeficiency Virus (HIV).
Bacterial Outer Membrane Proteins↗
High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation.
Salmonella typhimurium and S. typhi were transformed with high efficiency by electroporation. Transformation efficiencies of up to 10(10) transformants per microgram of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a ga1E mutation slightly improved transformation efficiency in S. typhimurium (less than tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.
Antigens, Bacterial↗
Immune responses in BALB/c mice following immunization with aromatic compound or purine-dependent Salmonella typhimurium strains.
Two near isogenic strains of Salmonella typhimurium HWSH, stably mutated in either the aroA gene affecting the biosynthesis of aromatic compounds, or the purA gene affecting the biosynthesis of purines, were administered intravenously as live attenuated vaccines to BALB/c mice. HWSH aroA-immunized mice were well protected against intravenous (i.v.) challenge with wild-type virulent HWSH for at least 10 weeks, whereas HWSH purA-immunized mice were unprotected. Furthermore, HWSH aroA-immunized mice could also control a heterologous challenge with virulent Listeria monocytogenes at 7 and 14 days post-immunization, whereas mice receiving a similar dose of HWSH purA could not. Increasing the i.v. dose of HWSH purA compared to HWSH aroA induced some resistance to L. monocytogenes. Induction of early anti-S. typhimurium resistance by HWSH aroA immunization appeared slightly later than the anti-L. monocytogenes resistance. Mice immunized with either vaccine were able to mount S. typhimurium-specific T-cell proliferative responses and produced anti-S. typhimurium humoral antibodies in their serum. The antibody titre was greater in those mice immunized with the aroA mutant.
Animals↗
Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. III. Delivery of recombinant nucleoprotein to the immune system using attenuated Salmonella typhimurium as a live carrier.
A plasmid encoding the influenza nucleoprotein gene from A/NT/60/68 virus was transduced into the attenuated Salmonella typhimurium aroA- strain SL3261. The bacterial vector expressing the viral gene product was able to induce both humoral and cell-mediated immune responses to the nucleoprotein antigen. CD4+ virus-specific T cells capable of proliferation were readily induced and, in some circumstances, class II major histocompatibility complex (MHC)-restricted cytotoxicity was detected. However, virus-specific class I MHC-restricted cytotoxic T lymphocytes (CTL) were not detected after such immunization. Mice immunized orally with the nucleoprotein-expressing bacteria mounted a strong anti-viral antibody response and spleen cells from such mice proliferated specifically to virus challenge in vitro, producing interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Orally immunized mice showed significant protection from challenge infection with influenza virus if the mice were also boosted intranasally before infection.
Animals↗
Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. II. Protection from influenza infection and mechanism of protection.
Protection against influenza A virus infection in mice immunized with recombinant nucleoprotein (rNP) was studied. Nucleoprotein-immune mice were protected against a lethal challenge with A/Puerto Rico/8/34 (A/PR8) virus but showed considerable morbidity before recovery. Local boosting of the immune system with rNP by intranasal immunization improved the protection in NP-immune mice, and the decrease in morbidity after infection was reflected in accelerated clearance of virus from lungs. However, immune, boosted mice also rapidly cleared an antigenically unrelated influenza B virus from their lungs. Mice immunized with rNP precipitated with alhydrogel, that subsequently developed significant resistance to virus infection, failed to generate detectable levels of class I major histocompatibility complex (MHC)-restricted killer cells. Furthermore, B10.A(5R) mice that are non-responders for NP-specific class I killer cells could also be protected by immunization with rNP. In contrast, rNP-immunized mice developed strong proliferative T-cell responses to rNP. These data argue for an important role for helper T cells rather than virus-specific class I cytotoxic T cells in protection against influenza virus infection induced by rNP.
Animals↗
Antiviral immunity induced by recombinant nucleoprotein of influenza A virus. I. Characteristics and cross-reactivity of T cell responses.
The gene for the nucleoprotein of the A/NT/60/68 influenza virus was expressed in bacteria and the recombinant protein purified. Lymph node cells from mice immunized with recombinant nucleoprotein proliferated in response to in vitro stimulation with a range of type A influenza viruses. Proliferation was inhibited by mAb to CD4 and class II MHC gene products. IFN-gamma was produced and type-specific CTL were generated in stimulated cultures of immune lymph node cells. These CTL were CD4+ and restricted to class II MHC gene products. Immunization with recombinant nucleoprotein generated Th cells in vivo as measured by the ability to generate an accelerated response to hemagglutinin after challenge with inactivated virus. The results are discussed with reference to a cross-reactive vaccine against influenza.
Animals↗
Oral vaccination.
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Administration, Oral↗
Construction and characterization of vaccine strains of Salmonella harboring mutations in two different aro genes.
Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harboring stable mutations in aroC alone or in aroC and aroA together. Fifty percent lethal doses after intravenous inoculation of the mutants into BALB/c mice were determined, and the mutants were as highly attenuated as were SL1344 aroA derivatives. All aro-dependent derivatives persisted in vivo at similar levels and for similar intervals in the livers and spleens of BALB/c mice infected intravenously. Mice vaccinated orally with 10(10) live organisms of the different derivatives survived and were well protected against oral challenge with virulent S. typhimurium. A Salmonella typhi Ty2 derivative, designated WBL2000, was constructed that harbors well-defined, stable mutations in both aroA and aroC.
3-Phosphoshikimate 1-Carboxyvinyltransferase↗