Biomedical subjects

D Baker

Publications and source records attributed to D Baker.

At least 163 records · Page 9Linked to original sources

PubMed1998

Financial difficulties, smoking habits, composition of the diet and birthweight in a population of pregnant women in the South West of England. ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood.

OBJECTIVE: To investigate the relationships between self-assessed financial difficulties, diet in pregnancy and birthweight. DESIGN: Information on diet composition, difficulty in affording food, smoking habits and parity was collected by self-completion questionnaires at 32 weeks of pregnancy. Information on birthweights was obtained from hospital records. SUBJECTS: A geographically defined population 11833 pregnant women resident in the South-West of England. MAIN OUTCOME MEASURES: Nutrient intakes, food choices and birthweight. RESULTS: Women with greater difficulty in affording food had lower intakes of protein, fibre, vitamin C, niacin, pyridoxine, iron, zinc, magnesium and potassium than did women with little or no difficulty. They were more likely to use cooking and spreading fats with a high saturates content, and less likely to eat fish, fruit, vegetables and salad. In a multivariate analysis including parity and smoking status, financial difficulty was found to have no significant relationship with birthweight. CONCLUSION: The quality of diet in pregnancy falls with increasing difficulty in affording food. However, this does not appear to affect mean birthweight of infants born after 32 weeks gestation.

Adolescent↗

PubMed1998

Cloning and expression of murine IFN beta and a TNF antagonist for gene therapy of experimental allergic encephalomyelitis.

Immunomodulation of an ongoing autoimmune disease can be achieved by inhibitory cytokines or cytokine inhibitors such as TNF antagonists, delivery by gene therapy. The aim of this study was to design and test plasmid and retrovirus vectors expressing the mouse IFN beta gene and a chimeric protein containing the extracellular domain of human p55 TNF receptor linked to a murine Ig. These vectors were transiently expressed in COS-7 cells and permanently in amphotropic packaging cell lines or ABH mouse immortalized fibroblasts. Expression levels were assessed by ELISA. Western blotting and biological activity. In order to achieve tissue-specific expression in the CNS, the IFN beta gene was cloned and expressed under the control of the rat NSE promoter. We evaluated these constructs by direct intracranial injections of DNA-liposome complexes during the induction phase of experimental allergic encephalomyelitis, a murine model of multiple sclerosis, with therapeutic benefit.

Animals↗

PubMed1998

Immune responses limit adenovirally mediated gene expression in the adult mouse eye.

In order to investigate the immunological consequences of gene transfer to the eye using viral vectors, adenovirus carrying a lacZ reporter gene (AV.LacZ) was injected either subretinally, subconjunctivally or into the anterior chamber of three groups of adult mice: immunocompetent or transiently immunosuppressed BALB/c mice and congenic immunodeficient nude mice. Adenovirally mediated lacZ expression persisted for approximately 3 weeks following injection of the vector into the anterior chamber, retina or extra ocular tissues of the conjuctiva of BALB/c mice. It appears that T cell-mediated immune responses limit the duration of AV-mediated ocular gene expression in adult mice since lacZ gene expression was detected for at least 15 weeks in T cell-deficient BALB/c nude mice, although the level of transgene expression decreased with time. Since intra-ocular AV-mediated gene expression was not significantly longer than extra-ocular expression, it appears that the eye is not normally immune-privileged with respect to viral vectors. Inflammatory cells were detected in the vitreous after anterior chamber injection and in the retina after subretinal injection of adenovirus. The presence of both CD4+ and CD8+ T cells was established by immunophenotyping. Reinjection of BALB/c mice resulted in rapid decline in reporter gene expression, but successful readministration was possible in the case of immunodeficient nude mice. However, after transient depletion of T cells, achieved by intraperitoneal injection of both CD8- and CD4-specific antibodies, the duration of expression in BALB/c mice was longer in the eye (at least 12 weeks, again with decrease in level over time), than in extra-ocular tissues (8 weeks) provided the animal was not reinjected with virus raising the possibility of partial ocular immune-privilege after transient immunosuppression.

Adenoviridae↗

PubMed1998

Co-injection of adenovirus expressing CTLA4-Ig prolongs adenovirally mediated lacZ reporter gene expression in the mouse retina.

There is growing interest in gene delivery to the eye in order to develop gene therapy for the many ocular disorders which may be amenable to this approach. To date, recombinant adenoviruses (AV) have been the main vector used for gene delivery to anterior and posterior segments in animal models. As with delivery to other organs, immune responses to vector and transgene limit the duration of expression in the eye. Using an E1-deleted adenoviral vector carrying a lacZ reporter gene, we have previously demonstrated that a T cell-mediated immune response reduces the level of intra-ocular transgene expression over time and limits it to around 3 weeks in mice. This report describes a strategy for prolonging gene expression by blocking the B7-CD28 interactions between antigen presenting cells (APC) and T cells in order to prevent the costimulatory signals required for T cell survival and proliferation. This was achieved by the co-injection of AV encoding a secreted immunomodulatory molecule (CTLA4-Ig) which consists of the extra-cellular domain of mouse CTLA4 fused to the Fc region of human IgG. Subretinal co-injection of AV encoding beta galactosidase with AV encoding CTLA4-Ig results in prolonged expression in retinal cells compared with subretinal injection of only adenovirus encoding beta galactosidase.

Abatacept↗

PubMed1998

Inequality in infant morbidity: causes and consequences in England in the 1990s. ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood.

STUDY OBJECTIVE: To examine the effect of deprivation, crowding, maternal smoking, and breast feeding on morbidity from wheeze and diarrhoea in the first six months after birth. DESIGN: A geographically located population survey using maternal responses on self completion questionnaires. SETTING: The three health districts of Bristol. SUBJECTS: 8501 infants from the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC) in which all women expecting a baby between April 1991 and December 1992 in Bristol were invited to participate. MAIN OUTCOME MEASURES: The prevalence and severity of wheeze at six months after birth. The prevalence of diarrhoea and the duration of the worst bout at six months after birth. RESULTS: Logistic regression analyses using a conceptual hierarchical framework showed that wheeze was significantly more likely to be reported for infants if they lived in rented accommodation (OR = 1.20, 95% CI = 1.04, 1.39), if they lived in crowded housing conditions (OR = 1.26, 95% CI = 1.06, 1.49), if they were one of a number of siblings (OR = 1.78, 95% CI = 1.52, 2.07), and if their mothers smoked (OR = 1.38, 95% CI = 1.21, 1.58). They were significantly less likely to have wheeze if they were breast fed (OR = 0.68, 95% CI = 0.59, 0.79). Each of these factors was independently related to the prevalence of wheeze. For infants with wheeze those who were breast fed for three or more months were significantly less likely to have three or more episodes in the first six months after birth (OR = 0.76, 95% CI = 0.58, 0.99). A higher prevalence of diarrhoea in infancy was associated with living in rented accommodation (OR = 1.25, 95% CI = 1.10, 1.41) and lower maternal education (OR = 0.76, 95% CI = 0.69, 0.84) and a lower prevalence with breast feeding (OR = 0.42, 95% CI = 0.37, 0.48). An episode of diarrhoea was significantly less likely to last for six or more days if an infant lived in mortgaged accommodation (OR = 1.34 95% CI = 1.03, 1.75) and was breast fed for three or more months (OR = 1.34 95% CI = 1.03, 1.75). CONCLUSION: Deprivation was associated with heightened morbidity from common conditions such as wheeze and diarrhoea for this geographical cohort of infants in England in the 1990s. Results supported evidence suggesting that breast feeding is protective against such conditions and is particularly associated with reduced severity and duration. Implications for future research and policy are discussed.

Breast Feeding↗

PubMed1998

The Importance of Heteroduplexes in Interpreting the Results of PCR-RED Diagnostic Assays: Application to the Analysis of Mutations in the Steroid 21-Hydroxylase Gene in a Case of Congenital Adrenal Hyperplasia.

Background: Congenital adrenal hyperplasia (CAH) due to deficiency of steroid 21- hydroxylase (CYP21) is an autosomal recessive disease that is a major cause of ambiguous genitalia at birth in females. The milder late-onset form of the disease can cause mild virilization in women including hirsutism, infertility, and acne. Characterization of the causative mutations in a patient requires finding mutations on both chromosomes. Methods and Results: We use polymerase chain reaction (PCR) followed by restriction enzyme digestion (RED), among several other methods, to detect mutations in the CYP21 gene. The authors found two different point mutations in a patient with CAH, V281L and Q318X. Did we find two mutations in trans which would account for the disease, or two mutations in cis with a still unknown mutation(s) on the other chromosome? We devised a method to determine the cis/trans nature of the mutations using PCR amplification, digestion with a restriction enzyme (ApaL I) diagnostic for V281L, gel purification of the mutant band which lacks the cutting site, digestion with a second restriction enzyme (Pst I) which detects Q318X, and gel electrophoretic analysis of the products. Although the results initially supported a a cis orientation, consideration of the impact of heteroduplex formation during the PCR on the products of RED showed that the orientation was actually trans. Conclusions: When DNA that is heterozygous for a mutation is amplified using the PCR, four double-stranded DNA products can be formed: the two homoduplexes and two heteroduplexes. The heteroduplex DNA must be considered to ensure correct interpretation of the results of PCR-RED analysis.

Journal Article↗

PubMed1998

Gene therapy for rheumatoid arthritis. Theoretical considerations.

Current understanding of the pathogenesis of rheumatoid arthritis has provided evidence that therapeutic benefit can be achieved by using antagonists targeted to the inflammatory cytokines involved, mainly tumour necrosis factor-alpha and interleukin-1. Gene delivery of antagonists, which can inhibit the production or action of these cytokines and other mediators, has been achieved in experimental animal models. This new method of delivery can produce therapeutic effects at lower concentrations and in a local environment, overcoming the adverse effects that often accompany protein therapy. However, several technological and biological restraints preclude the immediate adaptation of this method to human treatment. Based on the experimental evidence, possible target therapeutic genes, cell types and vector systems that could be used are discussed in this article.

Arthritis, Rheumatoid↗

PubMed1998

Measurement of active coagulation factors in Autoplex-T with colorimetric active site-specific assay technology.

Autoplex-T is a partially activated prothrombin complex concentrate used primarily for the treatment of patients expressing factor VIII inhibitors. While Autoplex-T has a demonstrated record of clinical effectiveness, the procoagulant composition of this material has not been reported. This absence of composition data is a reflection of the lack of techniques appropriate for accurately measuring an individual protease such as factor IXa in complex mixtures of similar proteases. The development of Colorimetric Active Site-Specific ImmunoAssay technology (CASSIA) has permitted the accurate analysis of the coagulant enzymes present in Autoplex-T. Ten lots of Autoplex-T were reacted with both biotinylated phenylalanylprolylarginine chloromethylketone and biotinylated glutamylglycylarginine chloromethylketone. Only activated forms of the clotting factors present in Autoplex-T react with the peptide chloromethylketones and were thus separated from the other proteins present in Autoplex-T by adsorption onto streptavidin. The individual proteins bound to streptavidin were then detected with specific antibodies. Mean results from the analysis of ten lots of Autoplex-T (mean values) are as follows: factor Xla, 5.9 nM or 0.8 microg/ml; factor Xa, 46.5 nM or 2.1 microg/ml; factor IXa, 177.8 nM or 11.7 microg/ml; factor VIIa, 68.6 nM or 3.3 microg/ml and factor IIa, 5.3 nM or 0.2 microg/ml. These results are discussed with respect to the mechanism of action of Autoplex-T in the treatment of factor VIII inhibitor patients.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Algorithm for nutritional support: experience of the Metabolic and Infusion Support Service of St. Jude Children's Research Hospital.

The Metabolic and Infusion Support Service (MISS) at St. Jude Children's Research Hospital was established in 1988 to improve the quality of nutritional support given to children undergoing therapy for cancer. This multidisciplinary group, representing each of the clinical services within the hospital, provides a range of services to all patients requiring full enteral or parenteral nutritional support. In 1991, the MISS developed an algorithm for nutritional support which emphasized a demand for a compelling rationale for choosing parenteral over enteral support in patients with functional gastrointestinal tracts. Compliance with the algorithm was monitored annually for 3 years, with full compliance defined as meeting all criteria for initiating support and selection of an appropriate type of support. Compliance rates were 93% in 1992, 95% in 1993 and 100% in 1994. The algorithm was revised in 1994 to include criteria for offering oral supplementation to patients whose body weight was at least 90% of their ideal weight and whose protein stores were considered adequate. Full support was begun if no weight gain occurred. Patients likely to tolerate and absorb food from the gastrointestinal tract were classified into groups defined by the absence of intractable vomiting, severe diarrhea, graft-vs.-host disease affecting the gut, radiation enteritis, strictures, ileus, mucositis and treatment with allogeneic bone marrow transplant. Overall, the adoption of the algorithm has increased the frequency of enteral nutritional support, particularly via gastrostomies, by at least 3-fold. Our current emphasis is to define the time points in therapy at which nutritional intervention is most warranted.

Algorithms↗

PubMed1997

Folding dynamics of the src SH3 domain.

The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4-3.7 M). Estimates of the free energy of unfolding, DeltaGUH2O, from guanidine denaturation, thermal denaturation, and kinetic experiments were in good agreement. To complement these data on the global characteristics of src SH3 folding, individual hydrogen-deuterium (HD) exchange rates were measured for approximately half of the backbone amides in 0 and 0.7 M GdmCl. The calculated free energies of the opening reaction leading to exchange (DeltaGHD) indicated that unfolding is highly cooperative--slowly exchanging protons were distributed throughout the core of the protein. The slowly exchanging protons exhibited DeltaGHD values higher than the global DeltaGUH2O by approximately 1 kcal/mol, suggesting that the denatured state might be somewhat compact under native conditions. Comparison of the src SH3 with homologous SH3 domains as well as with other small well-characterized beta-sheet proteins provides insights into the determinants of folding kinetics and protein stability.

Amino Acid Sequence↗

PubMed1997

Contrasting roles for symmetrically disposed beta-turns in the folding of a small protein.

To investigate the role of turns in protein folding, we have characterized the effects of combinatorial and site-directed mutations in the two beta-turns of peptostreptococcal protein L on folding thermodynamics and kinetics. Sequences of folded variants recovered from combinatorial libraries using a phase display selection method were considerably more variable in the second turn than in the first turn. These combinatorial mutants as well as strategically placed point mutants in the two turns had a similar range of thermodynamic stabilities, but strikingly different folding kinetics. A glycine to alanine substitution in the second beta-turn increased the rate of unfolding more than tenfold but had little effect on the rate of folding, while mutation of a symmetrically disposed glycine residue in the first turn had little effect on unfolding but slowed the rate of folding nearly tenfold. These results demonstrate that the role of beta-turns in protein folding is strongly context-dependent, and suggests that the first turn is formed and the second turn disrupted in the folding transition state.

Bacterial Proteins↗

PubMed1997

Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability.

The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of 1/T on constant stability contours in the temperature-denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed.

Kinetics↗

PubMed1997

The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma.

BACKGROUND: It has been hypothesized that asthma-related health problems are most severe among children in inner-city areas who are allergic to a specific allergen and also exposed to high levels of that allergen in bedroom dust. METHODS: From November 1992 through October 1993, we recruited 476 children with asthma (age, four to nine years) from eight inner-city areas in the United States. Immediate hypersensitivity to cockroach, house-dust-mite, and cat allergens was measured by skin testing. We then measured major allergens of cockroach (Bla g 1), dust mites (Der p 1 and Der f 1), and cat dander (Fel d 1) in household dust using monoclonal-antibody-based enzyme-linked immunosorbent assays. High levels of exposure were defined according to proposed thresholds for causing disease. Data on morbidity due to asthma were collected at base line and over a one-year period. RESULTS: Of the children, 36.8 percent were allergic to cockroach allergen, 34.9 percent to dust-mite allergen, and 22.7 percent to cat allergen. Among the children's bedrooms, 50.2 percent had high levels of cockroach allergen in dust, 9.7 percent had high levels of dust-mite allergen, and 12.6 percent had high levels of cat allergen. After we adjusted for sex, score on the Child Behavior Checklist, and family history of asthma, we found that children who were both allergic to cockroach allergen and exposed to high levels of this allergen had 0.37 hospitalization a year, as compared with 0.11 for the other children (P=0.001), and 2.56 unscheduled medical visits for asthma per year, as compared with 1.43 (P<0.001). They also had significantly more days of wheezing, missed school days, and nights with lost sleep, and their parents or other care givers were awakened during the night and changed their daytime plans because of the child's asthma significantly more frequently. Similar patterns were not found for the combination of allergy to dust mites or cat dander and high levels of the allergen. CONCLUSIONS: The combination of cockroach allergy and exposure to high levels of this allergen may help explain the frequency of asthma-related health problems in inner-city children.

Allergens↗

PubMed1997

Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions.

We explore the ability of a simple simulated annealing procedure to assemble native-like structures from fragments of unrelated protein structures with similar local sequences using Bayesian scoring functions. Environment and residue pair specific contributions to the scoring functions appear as the first two terms in a series expansion for the residue probability distributions in the protein database; the decoupling of the distance and environment dependencies of the distributions resolves the major problems with current database-derived scoring functions noted by Thomas and Dill. The simulated annealing procedure rapidly and frequently generates native-like structures for small helical proteins and better than random structures for small beta sheet containing proteins. Most of the simulated structures have native-like solvent accessibility and secondary structure patterns, and thus ensembles of these structures provide a particularly challenging set of decoys for evaluating scoring functions. We investigate the effects of multiple sequence information and different types of conformational constraints on the overall performance of the method, and the ability of a variety of recently developed scoring functions to recognize the native-like conformations in the ensembles of simulated structures.

Bayes Theorem↗

PubMed1997

Kinetics of folding of the IgG binding domain of peptostreptococcal protein L.

The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins.

Antigen-Antibody Reactions↗

PubMed1997

Blind predictions of local protein structure in CASP2 targets using the I-sites library.

Blind predictions of the local structure of nine CASP2 targets were made using the I-sites library of short sequence--structure motifs, revealing strengths and weaknesses in this new knowledge-based method. Many turns between secondary structural elements were accurately predicted. Estimates of the confidence of prediction correlated well with the accuracy over the whole set. Bias toward structures used to develop the library was minimal, probably because of the extensive use of cross-validation. However, helix positions were better predicted by the PHD program. The method is likely to be sensitive to the quality of the sequence alignment. A general measure for evaluating local structure predictions is suggested.

Binding Sites↗

PubMed1997

Local interactions and the optimization of protein folding.

The role of local interactions in protein folding has recently been the subject of some controversy. Here we investigate an extension of Zwanzig's simple and general model of folding in which local and nonlocal interactions are represented by functions of single and multiple conformational degrees of freedom, respectively. The kinetics and thermodynamics of folding are studied for a series of energy functions in which the energy of the native structure is fixed, but the relative contributions of local and nonlocal interactions to this energy are varied over a broad range. For funnel shaped energy landscapes, we find that 1) the rate of folding increases, but the stability of the folded state decreases, as the contribution of local interactions to the energy of the native structure increases, and 2) the amount of native structure in the unfolded state and the transition state vary considerably with the local interaction strength. Simple exponential kinetics and a well-defined free energy barrier separating folded and unfolded states are observed when nonlocal interactions make an appreciable contribution to the energy of the native structure; in such cases a transition state theory type approximation yields reasonably accurate estimates of the folding rate. Bumps in the folding funnel near the native state, which could result from desolvation effects, side chain freezing, or the breaking of nonnative contacts, significantly alter the dependence of the folding rate on the local interaction strength: the rate of folding decreases when the local interaction strength is increased beyond a certain point. A survey of the distribution of strong contacts in the protein structure database suggests that evolutionary optimization has involved both kinetics and thermodynamics: strong contacts are enriched at both very short and very long sequence separations.

Kinetics↗

PubMed1997

A deletion polymorphism due to Alu-Alu recombination in intron 2 of the retinoblastoma gene: association with human gliomas.

The retinoblastoma gene (RB) encodes a tumor suppressor that is inactivated in a number of different types of cancer. We searched for gross alterations of this gene in tumors of the central nervous system by using Southern blot hybridization. A common alteration was found in several tumors and was mapped to the region around exon 2. Nucleotide sequencing showed that the alteration was caused by a 799-bp deletion in intron 2 of the RB gene and was probably due to homologous recombination between two Alu repeats. Deletions of this type have not been found previously in the RB gene. The deletion turned out to be a polymorphism with an allele frequency estimated at 2.2% in 185 patients without cancer. The deletion was found in five of 48 patients with brain tumors (allele frequency of 5.2%). This difference is not statistically significant (P = 0.149, Fisher's exact test). Confining the analysis only to glioma brain tumors revealed a statistically significant difference compared with the cancer-free patient controls (P = 0.027, Fisher's exact test). Further study is needed to determine if the deletion is a weak brain cancer-predisposing mutation or a harmless polymorphism. Finding this mutation in a tumor and the germline DNA of a retinoblastoma patient could lead to incorrect estimation of the heritability of a tumor.

Base Sequence↗