BSE coordinator should have been full-time job.
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Animals↗
Biomedical subjects
F Brown
Publications and source records attributed to F Brown.
Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket.
A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9-fold), in vitro motility (2.7-fold), and the Vmax of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.
Actins↗
A universal virus inactivant for decontaminating blood and biopharmaceutical products.
Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. For biopharmaceutical products, removal can usually be achieved by a series of fractionation steps or by inactivation with a suitable reagent. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Viruses belonging to all the recognised families can be inactivated by imines. In this paper it is shown that the biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Also shown is that both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The RNA extracted from FMDV inactivated at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region. Surprisingly, it could be translated in the rabbit reticulocyte system although some of the products were different from those obtained with unmodified RNA.
Animals↗
Pharmacokinetic interactions between two human immunodeficiency virus protease inhibitors, ritonavir and saquinavir.
OBJECTIVE: To assess the pharmacokinetic interaction between ritonavir and saquinavir. METHODS: Ritonavir and saquinavir were administered in single doses to six groups of healthy volunteers in a two-way (saquinavir alone and ritonavir plus saquinavir for groups I through V) and a three-way (ritonavir alone, saquinavir alone, and ritonavir plus saquinavir for group VI) crossover manner with the following doses: group I, 200 mg saquinavir and 300 mg ritonavir; group II, 200 mg saquinavir and 600 mg ritonavir; group III, 400 mg saquinavir and 300 mg ritonavir; group IV, 400 mg saquinavir and 600 mg ritonavir; group V; 600 mg saquinavir and 200 mg ritonavir; group VI, 600 mg saquinavir and 600 mg ritonavir. RESULTS: Coadministration of ritonavir markedly increased the area under the plasma concentration-time curve (AUC) and peak concentration of saquinavir (> 50-fold and 22-fold, respectively). For a constant ritonavir dose, the pharmacokinetics of saquinavir were relatively proportional to dose. For a constant saquinavir dose, the increase in saquinavir concentration tended to be less than proportional to ritonavir dose. Ritonavir reduced intersubject variability in the saquinavir AUC from 60% to 28%. The in vivo inhibition constant was 0.025 +/- 0.020 micrograms/ml with noncompartmental estimation and 0.0164 +/- 0.0004 micrograms/ml with nonlinear mixed-effects model compartmental analysis. Saquinavir showed no clinically significant effect on the pharmacokinetics of ritonavir (+6.4% in AUC). The regimens were well tolerated. CONCLUSIONS: The large effect of ritonavir on the pharmacokinetics of saquinavir is consistent with a large reduction of saquinavir first-pass metabolism and postabsorptive clearance. Given the limited bioavailability of saquinavir given in the hard gelatin capsule formulation, this drug interaction is expected to have implications in the use of protease inhibitors in the management of human immunodeficiency virus infection.
Adult↗
The use of pharmacoscintigraphy to elucidate food effects observed with a novel protease inhibitor (saquinavir).
PURPOSE: To evaluate mechanistically the effect of food on the absorption and gastrointestinal transit of the protease inhibitor saquinavir. METHODS: Pharmacoscintigraphic investigation in eight healthy volunteers. RESULTS: Gastric emptying occurred rapidly in the fasted state with some capsules leaving the stomach prior to disintegration. Unmeasurable plasma concentrations were observed in several subjects when dosed under fasted conditions. Following post-prandial administration the radioactive marker became re-distributed within the stomach contents and consequently slower gastric emptying resulted. Plasma concentrations under fed conditions were measurable up to 12 hrs after administration in seven of the eight subjects. Six of the eight plasma profiles showed secondary peaks at c. 4 hours post-dose; two of which coincided with the gastrocolonic response following ingestion of lunch. CONCLUSIONS: Bioavailability of saquinavir is significantly improved in the presence of food. Emptying of intact capsules in the fasted state may further reduce bioavailability. In the fed state, capsules disintegrate rapidly and gastric emptying is prolonged which may improve exposure of the drug to target absorption sites. Saquinavir may be absorbed from the colon. Second peaks in the absorption profile can only be attributed to gastrocolonic response following ingestion of a meal in some cases. Increased absorption is more likely to be due to an increase in dissolved drug being available for absorption due to general increased motility and secretion stimulated by ingestion of a meal.
Adult↗
Impact of erythromycin on respiratory colonization of Ureaplasma urealyticum and the development of chronic lung disease in extremely low birth weight infants.
BACKGROUND: Chronic lung disease (CLD) is a significant cause of neonatal morbidity and mortality despite advances in neonatal care. Ureaplasma urealyticum colonization of the lower respiratory tract has been associated with CLD, particularly in extremely low birth weight infants. Despite numerous studies demonstrating the pathogenicity of this organism, treatment remains controversial. This study examines neonates colonized with U. urealyticum in the lower respiratory tract and treated with erythromycin, as compared with noncolonized neonates. METHODS: A prospective cohort study of 124 neonates weighing <1000 g at birth, requiring endotracheal intubation and ventilation. Endotracheal aspirates were cultured for U. urealyticum and conventional bacteria twice weekly for the duration of endotracheal intubation. Infants colonized with U. urealyticum were treated with intravenous erythromycin. Maximal ventilatory requirements, CLD at Day 28 and 36 weeks postconception, duration of ventilation, oxygen dependency and hospital stay were documented. RESULTS: Twenty-two infants (18%) were identified as being U. urealyticum colonized in endotracheal aspirates. Colonization was significantly associated with younger maternal age, prolonged rupture of membranes, premature labor and vaginal delivery. Of colonized neonates 14% were delivered by cesarean section, with intact membranes. As compared with noncolonized infants, there were no statistically significant differences in chronic lung disease, duration of oxygen therapy or time to discharge. CONCLUSIONS: Seven published cohort studies of similar high risk populations where U. urealyticum-colonized infants did not receive erythromycin therapy, show a consistent association with CLD (pooled relative risk + 5.21; 95% confidence interval, 2.93 to 9.64). This association was not demonstrated in the current study and adds further weight to the need for a randomized controlled trial to be performed to evaluate this treatment regimen.
Anti-Bacterial Agents↗
The potential of retro-inverso peptides as synthetic vaccines.
Retro-inverso peptides, also known as all-D-retro or retro-enantio peptides, are composed of D-amino acids assembled in the reverse order from that of the parent L-sequence. Since the orientation of the side-chains in a retro-inverso analogue is very similar to that in the parent L-peptide, this leads to a high level of antigenic cross-reactivity between the two peptides. The potential of retro-inverso peptides as synthetic vaccines has been investigated in the case of foot-and-mouth disease. A single inoculation of retro-inverso peptide corresponding to residues 141-159 of the VP1 protein of foot-and-mouth disease virus induced longer-lasting and higher antibody titres in immunised animals than the corresponding L-peptides. The antibodies cross-reacted strongly with virus particles and with L-peptides and conferred substantial protection in guinea-pigs challenged with the cognate virus. Retro-inverso peptides have considerable potential as synthetic vaccines, since their increased resistance to proteases may overcome one of the major drawbacks of classical L-peptide vaccines.
Journal Article↗
The potential of retro-inverso peptides as synthetic vaccines.
Retro-inverso (RI) peptides, also called all-D-retro peptides, have been shown to mimic the antigenic and immunogenic properties of L-peptides successfully. RI peptides corresponding to the loop 141-159 of the VP1 protein of foot-and-mouth disease virus (FMDV) have been synthesized and used to immunize rabbits and guinea pigs. These peptides induced longer-lasting and higher antibody titres in immunized animals than did the corresponding L-peptides and the antibodies cross-reacted strongly with virus particles and with L-peptides. Antisera raised to RI peptides had in vitro virus neutralization titres equal to or better than those obtained after immunization with classical FMDV antigens and L-peptides. In view of their increased stability, RI peptides may overcome some of the shortcomings of synthetic viral vaccines based on L-peptides.
Amino Acid Sequence↗
Differentiating foot-and-mouth disease virus-infected from vaccinated animals with baculovirus-expressed specific proteins.
We had shown in preliminary studies with a small number of animals that antibodies against 2C could be detected in cattle and pigs which had been infected with FMDV but not in animals which had been vaccinated against the disease. To determine whether this test was generally applicable, sera from several hundred animals which had been vaccinated with different products in many countries have been tested in an ELISA using baculovirus expressed 2C. Our results show that only 1-2% of the sera gave a positive reaction by this method. In contrast, 100% of sera from convalescent animals gave a positive reaction. To be useful in differentiating between convalescent and vaccinated animals it is necessary to know how long these antibodies can be detected by our ELISA. We have determined the levels of antibodies against 2C and also other virus-specific proteins which are present in cattle and pigs following infection with FMDV. Our results show that levels of anti-3ABC antibodies could be detected by ELISA with baculovirus-expressed protein up to one year after infection. In contrast, the levels of anti-2C antibodies fell more rapidly than those against 3ABC indicating that the latter protein may be preferable for detecting convalescent animals. Nevertheless, we envisage that the final test format should include several virus-specific proteins to determine accurately the immune status of an animal.
Animals↗
Cattle response to foot-and-mouth disease virus nonstructural proteins as antigens within vaccines produced using different concentrations.
Four groups of ten nine-month-old Nelore heifers were used for this study. Each group received one of four foot-and-mouth disease (FMD) trivalent vaccines for the duration of the experiment. The four vaccine formulations (Normal, 2X, 4X and 8X) differed in 140S content to determine the serological reactivities to FMD virus (FMDV) nonstructural proteins 2C, 3ABC and 3D. Vaccination was by the intramuscular administration of vaccine on day 0, 180 and 360. Bleedings were done at 30 days post vaccination (dpv), 90 dpv, 30 days post revaccination (dpr), 90 dpr, and 30 days post third administration (dprr). There was a general tendency to have higher mean 3D responses with increased vaccine application but not with increased concentration of antigen. With 2C and 3ABC this tendency was not seen, neither with repeated application of vaccine nor with increased antigen concentration. All individual animal observations to 2C and 3ABC remained within three standard deviations of the average observed for naive bovids. Percent of positive (PP) reactions was determined using an ELISA for nonstructural proteins 2C, 3ABC and 3D expressed in baculovirus as previously described. A value of > 25 PP to 2C or 3ABC could be considered as an indication of previous infection or of the presence of viral activity. PP results between 18 and 25 PP suggest viral activity and animals should be retested. Those responses below 15 PP are suggestive of vaccination or naive status. As diagnosis in the laboratory is not divorced from the field epidemiological scene, the intermediate zone between 10 and 20 PP should be considered and acted upon according to the overall zoosanitary situation of that country or region and the purposes of the ongoing FMD control efforts.
Animals↗
Problems with BHK 21 cells.
The properties of baby hamster kidney (BHK 21) cells are modified by passage in suspension culture. The suspended cells differ from monolayer cells in the surface expression of some integrin chains involved in attachment of foot-and-mouth disease virus (FMDV), in particular the progressive down-regulation of both alpha5 and alphaV integrin chains. This down-regulation is correlated with the loss of actin stress fibres. FMDV particles from these cells are unstable towards the aziridine used in inactivating the virus for vaccine production. Moreover, growth of virus in suspended cells can lead to the selection of antigenic variants which differ from those produced in monolayer cells.
Animals↗
Design of [R-(Z)]-(+)-alpha-(methoxyimino)-1-azabicyclo[2.2.2]octane-3-acetonitri le (SB 202026), a functionally selective azabicyclic muscarinic M1 agonist incorporating the N-methoxy imidoyl nitrile group as a novel ester bioisostere.
Loss of cholinergic function is believed to be implicated in the cognitive decline associated with senile dementia of the Alzheimer type (SDAT). The disease is characterized by progressive loss of muscarinic receptors located on nerve terminals while postsynaptic muscarinic M1 receptors appear to remain largely intact. Muscarinic agonists acting directly on postsynaptic receptors offer the prospect of countering the cholinergic deficit in SDAT. This study describes a novel series of azabicyclic muscarinic agonists, which incorporate an oxime ether or modified oxime ether group as an ester bioisostere. Modification of the oxime ether function by the introduction of electron withdrawing groups led to the finding that the (Z)-N-methoxy imidoyl nitrile group serves as a stable methyl ester bioisostere. This culminated in the discovery of the quinuclidinyl N-methoxy imidoyl nitrile R-(+)-(Z)-5g which is a functionally selective muscarinic M1 partial agonist currently in phase III clinical trials for the treatment of SDAT. The selective profile of R-(+)-(Z)-5g can be rationalized in terms of the relative affinity of the compound at muscarinic receptor subtypes, the degree of agonist efficacy, and brain penetrancy.
Alzheimer Disease↗
A retro-inverso peptide corresponding to the GH loop of foot-and-mouth disease virus elicits high levels of long-lasting protective neutralizing antibodies.
Peptides corresponding to the immunodominant loop located at residues 135-158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141-159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-D retro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the corresponding L-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the corresponding L-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines.
Amino Acid Sequence↗
The recognition of haemoglobin by antibodies raised for the immunoassay of beta-amyloid.
Canine and porcine cerebrospinal fluid (CSF) were fractionated by size exclusion chromatography and analysed by a luminescence enzyme linked immunosorbent assay (ELISA) configured to detect beta-amyloid. A peak of activity was observed in the CSF consistent with the molecular weight of beta-amyloid. When CSF contaminated with blood was analysed an additional peak of immunoreactivity at a higher molecular weight was observed. The peak of activity was found to be derived from cross-reactivity of the immunoglobulins employed in the ELISA with haemoglobin. These findings are discussed with reference to primary and structural sequence homology between beta-amyloid and haemoglobin from a number of species, the known properties of beta-amyloid and recent clinical reports.
Amyloid beta-Peptides↗
Structural comparison between retro-inverso and parent peptides: molecular basis for the biological activity of a retro-inverso analogue of the immunodominant fragment of VP1 coat protein from foot-and-mouth disease virus.
Antibodies induced against intact foot-and-mouth disease Virus (FMDV) particles bind to the retro-inverso analogue of fragment 141-159 of the viral coat protein VP1 of FMDV, variant A, equally well as to the parent peptide. A conformational investigation of this retro-inverso peptide was carried out by nmr spectroscopy and restrained molecular modeling in order to identify the structural basis for the antigenic mimicry between these retro-inverso and parent peptides. In 100% trifluoroethanol a well-defined left-handed alpha-helical region exists from residue 150 to residue 159, which is consistently present in all conformational families obtained from restrained modelling. A less-defined left-handed helical region is present in the tract 144-148, which is also consistent for all structures. Conformational flexibility exists about Gly149, which leads to two types of structures, either bent or linear. In the bent structures, a three-residue inverse tight turn is found, which can be classified as an inverse gamma-turn centered at Gly149. The overall structural features of the retro-inverso peptide are shown to be similar to those of the parent L-peptide. The two molecules, however, are roughly mirror images because they share inherently chiral secondary structure elements. By comparing these conformational conclusions with the x-ray structure of the Fab complex of a corresponding VP1 antigenic fragment, a rationale is proposed to account for the topological requirements of specific recognition that are implied by the equivalent antigenic activity of the natural and retro-inverso compounds.
Amino Acid Sequence↗
Role and regulation of phospholipase D activity in normal and cancer cells.
PLD is regulated by the small GTP binding proteins Rho and Arf, though predominantly by the latter. The PA product of PLD activation is an activator of Rho-regulated actin stress fibre formation and in invasive cells of MMP-9 synthesis and activation. Together this may explain the increased invasion of cells in response to PA.
ADP-Ribosylation Factors↗
Baculovirus expressed 2C of foot-and-mouth disease virus has the potential for differentiating convalescent from vaccinated animals.
Determining whether animals have been infected with foot-and-mouth disease virus or vaccinated is important because infected animals frequently become carriers of the virus, shed it intermittently and thus may be the source of new outbreaks of the disease. We had shown previously that the sera of convalescent animals contain antibodies to 2C, a highly conserved non-structural protein, whereas the sera of vaccinated animals do not. This is explained by observation that 2C is retained on the membranes of cells used for growing the virus for vaccine production. In contrast, the non-structural protein 3D, which is released into the medium, is not removed by centrifugation or filtration during vaccine production and therefore stimulates an immune response in both vaccinated and convalescent cattle. In this study we produced 2C and 3D in insect cells infected with recombinant baculoviruses. As demonstrated by serology and electron microscopy, 2C is also retained on the membranes of the insect cells. Both expressed proteins react with sera of convalescent animals, indicating that they are conformationally similar, but the 2C does not react with sera from vaccinated animals. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test.
Animals↗
Empirically validated family interventions for pediatric psychology: sickle cell disease as an exemplar.
Prompted by the growth of managed care and the American Psychological Association's recent guidelines for treatment efficacy studies, we have struggled with the challenges associated with devising and implementing manualized family intervention programs for pediatric patients. This manuscript outlines the strengths and challenges of controlled manual-based family interventions for medically ill children, using pediatric sickle cell disease (SCD) as an example. A culturally and developmentally sensitive intervention program, designed for the researcher's subject population (African American, low SES, inner city) is discussed. Possible solutions to the challenges of conducting family-oriented intervention efficacy studies with pediatric populations are presented.
Adolescent↗