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Biomedical subjects

A F Johnson

Publications and source records attributed to A F Johnson.

At least 19 recordsLinked to original sources
PubMed1996

Fluorescence-based sequencing of double-stranded DNA by hexamer string priming.

A fluorescence-based, T7 (Sequenase) dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded binding protein is described. In this method, the circular, supercoiled DNA vector pUC19 is first linearized with a restriction enzyme to create a sequenceable template. Sequencing is then accomplished using three cycles of "denaturation," annealing, and extension/termination. Twenty-two of 33 hexamer strings tested in a controlled study produced acceptable sequence, with read lengths varying from the mid 300s to the low 400s and a base-calling accuracy of at least 97%. To test its potential utility in directed DNA sequencing, the protocol was then used to completely sequence both strands of pUC19. For this test project, a total of 28 hexamer strings was used with an overall successful priming rate of 75%. The current protocol appears to be sufficiently robust to be used in the finishing phase of a shotgun sequencing project and is amenable to semiautomation. Prospects for using the protocol for full-scale directed sequencing as well as for full automation are discussed.

Automation↗
PubMed1996

M13-102: a vector for facilitating construction and improving quality of M13 shotgun libraries.

A modified vector, M13-102, is described which utilizes the previously reported M13-100 direct selection strategy for shotgun cloning [Guilfoyle and Smith, Nucleic Acids Res. 22 (1994) 100-107]. In these vectors, direct selection replaces the need for phosphatase treatment of vector DNA and is achieved by insertional inactivation of M13 gene X. When not inactivated, the engineered overproduction of the M13 gene X product mediates phage replication repression. M13-102 contains two new additions: (1) a sequence enabling triple-helix-mediated affinity capture (TAC) for purification of linearized vector DNA, and (2) universal primer sequences for wider compatibility with commercial instruments that support fluorescence-based sequencing. Using a biotinylated homopyrimidine oligodeoxyribonucleotide as third-strand probe, TAC is performed on streptavidin-coated magnetic beads [Ji et al., Genetics Analysis: Techniques and Applications 11 (1994) 43-47], and serves as a rapid and efficient alternative to gel purification. To reduce tandem insertions, phosphatase treatment of insert DNA was easily invoked without sacrificing cloning efficiency. The combined capabilities of direct selection, TAC purification and phosphatase treatment of inserts should facilitate library construction and improve overall library quality.

Bacteriophage M13↗
PubMed1996

Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture.

A solid-phase triple-helix-mediated affinity capture method is described for the purification of single-stranded M13 DNA for use as template in fluorescence-based DNA sequencing reactions. In this method, a biotinylated polypyrimidine oligonucleotide "loop" bound to streptavidin-coated magnetic beads is used to selectively capture single-stranded M13 DNA from high-titer phage supernatant through the formation of a cooperative triple helix (CTH) complex between the oligonucleotide and a polypurine site previously cloned into the M13 vector. Capture is accomplished at acidic pH to encourage triple-helix formation, while elution is performed at alkaline pH with heating to destroy the CTH complex. The beads can be reused up to three times without probe replenishment. Yields of M13 ssDNA in excess of 1 microgram per milliliter of culture are obtained, sufficient for use as template in fluorescence-based DNA sequencing reactions.

Bacteriophage M13↗
PubMed1993

Nonisotopic DNA detection system employing elastase and fluorogenic rhodamine substrate.

An alternative fluorescence-based method has been developed for the direct detection of small quantities of DNA in solution. In this system, a serine protease (elastase) is coupled to a DNA oligonucleotide through a disulfide linkage. A bis-(tetraalanine)-derivatized rhodamine molecule BZTAlaR) has been synthesized for use as a substrate. BZTAlaR is nonfluorescent in its derivatized form and shows negligible hydrolysis in solution. Cleavage of the tetraalanyl groups from the rhodamine portion of the molecule restores its fluorescence. Hybridization of the elastase-oligonucleotide conjugate to its target, capture of the conjugate-target complex with streptavidin-coated magnetic beads, addition of substrate, and subsequent detection of the target by fluorescence are accomplished in solution. Hybridization is rapid and specific, with over 90% of a target sequence successfully hybridized and captured. This method exhibits low background and an amplified fluorescent signal over time, resulting in a current detection limit of 0.49 fmol of elastase alone, or 2.64 fmol of conjugate, within 2 h.

Base Sequence↗
PubMed1991

Optical isomers of rocastine and close analogues: synthesis and H1 antihistaminic activity of its enantiomers and their structural relationship to the classical antihistamines.

The enantiomers of 2-[2-(dimethylamino)ethyl]-3,4-dihydro-4-methylpyrido[3,2-f]-1,4- oxazapine-5(2H)-thione (rocastine) and two of its more potent analogues were prepared with an enantiomeric purity of greater than 99.9%. The antihistaminic activity of these compounds was assessed by their ability to block histamine-induced lethality in guinea pigs and to inhibit [3H]mepyramine binding to guinea pig cortex. In this series, compounds having the R configuration at the 2-position are at least 300 times more potent than the S isomers. Conformational analysis and molecular modeling suggest that rocastine can adopt a conformation in which the pyridine ring, ether oxygen, and protonated amine functions are positioned similarly to the corresponding elements of the probable binding conformers of some of the more classical antihistamines. This conformation, boatlike in the oxazepine ring with the side chain quasi-equatorial and folded back toward the ring, is the likely binding conformer at the histamine H1 receptor, and the available structure-activity relationship data is consistent with this interpretation.

Animals↗
PubMed1990

Management of voice disorders.

Voice disorders are commonly seen in general medical practice. In some cases voice disorders represent the presenting symptom for serious underlying disease. It is important for clinicians from internal medicine, pediatrics, and family practice to be able to identify those factors in the history or observed vocal symptoms which suggest need for referral for comprehensive voice evaluation as well as to understand the distinct but complementary roles of the specific disciplines (otolaryngology and speech-language pathology) involved in diagnosis and treatment of patients with voice disorders.

Humans↗
PubMed1989

Benzo- and pyrido-1,4-oxazepin-5-ones and -thiones: synthesis and structure-activity relationships of a new series of H1 antihistamines.

A series of novel benzo- and pyrido-1,4-oxazepinones and -thiones which represents a new structural class of compounds possessing H1 antihistaminic activity was synthesized, and the SARs were evaluated. The antihistaminic activity was determined by blockade of histamine-induced lethality in guinea pigs. The sedative potential was determined by comparison of the EEG profiles of the compounds with those of known sedating and nonsedating antihistamines. Several of the compounds were shown to possess potent H1 antihistaminic activity and to be free of the cortical slowing with synchronized waves and spindling activity found in the EEG of sedative antihistamines. One compound, 2-[2-(dimethylamino)ethyl]-3,4-dihydro-4-methylpyrido[3,2-f]-1,4- oxazepine-5(2H)-thione (rocastine) is currently undergoing clinical evaluation as a nonsedating H1 antihistamine.

Animals↗
PubMed1986

Benzo-pyrones in the treatment of chronic schizophrenic diseases.

Sixteen chronic schizophrenic subjects were treated for 3 months each with either a benzo-pyrone (Paroven/Venoruton, Zyma) or a placebo in a randomized, double-blind crossover trial. They continued to take their previous drug therapies. Therapeutic effects were measured by the Brief Psychiatric Rating Scale (BPRS), by self-assessment, and by assessment by a relative. Eleven patients completed the trial in relation to BPRS assessment. When on the active substance, as compared with the placebo, they showed a mean improvement of 27% (significant at the 1% level). Ten patients completed the trial in relation to the self and relative's assessments. There were improvements of 16% and 13%, respectively (significant at the 5% levels). Half the patients showed improvements on all the tests. Their improvements were 49%, 31%, and 21%, respectively. There was evidence that the active substance began to have an effect within 2 weeks. No side effects were observed with the active substance.

Adult↗
PubMed1984

Psycho-social achievement in the latency-aged child with spina bifida within the family structure.

This project aims: 1) to appraise the psycho-social development of a sample of latency-aged spina bifida children upon entry to school; 2) to learn about the social influences that promote or impede the child's development, and 3) to identify ways that psycho-social intervention can be useful to families in which there is a congenitally disabled child. A longitudinal, observational case study was conducted over a six month period. This approach provided information on the impact of social influences, disability, and trauma on the child's development. The results indicate that a delay in reaching the important physical milestones of independent locomotion and continence hinders the achievement of standard psycho-social developmental goals; it does not exclude their achievement. By the sixth month in first grade, all of the children had adapted satisfactorily to school. Thus, the difference between the able-bodied and the spina bifida child was not one of kind, but one of degree. Spina bifida was found to put no ceiling on the child's psycho-social development.

Achievement↗
PubMed1982

A technique for identifying the subgroup membership of certain misarticulating children.

Cluster analysis was used to identify two homogeneous clusters of 8-9 1/2-year-old children who misarticulated /s/, /r/, or both. The analysis was based on the children's scores on 40 measures of language, reading, auditory processing, and other variables. Discriminant function analysis was then used to identify a subset of five measures and a means of computing classification scores. These measures and the classification scores can be used to identify the cluster membership of new subjects. The use of classification scores for identifying cluster membership was cross-validated against cluster analysis of a second group of children. The two clusters are described in terms of their performance on language and reading measures.

Articulation Disorders↗
PubMed1978

Assessment of parent-administered listening training for preschool children with articulation deficits.

Two studies concerning preschool misarticulating children are reported. The first study was concerned with direct effects of two varieties of parent-administered listening training. The second study focused on the influence of that same training on children's responses to sound-production training. Subjects were assigned to one of three conditions: listening, reading-talking, and control. Children in the first group were provided training by their parents that was intended to focus the child's attention on consonants in syllables or words and to teach discrimination between correctly and incorrectly articulated consonants. Parents of children in the second group read to and talked with them about the material. Neither treatment group surpassed the control group in gains made on any auditory processing or articulatory measure employed, and some parents found the listening training frustrating. In the second study, a subset of the children in each of the three groups was given sound-production training. The data obtained did not show any effect from earlier listening experiences on sound-production performance.

Attention↗
PubMed1977

Identification and description of homogeneous subgroups within a sample of misarticulating children.

A multivariate, hierarchical clustering procedure was used to identify homogeneous subgroups among 98 misarticulating children age eight years through nine years, six months. Clustering was based on 40 tests or measures of language, auditory processing, school achievement, oral structure, oral form recognition, and other phenomena. Clusters were described and then compared for performance on measures of articulation status and articulation improvement with training. Clusters were also derived from subsets of children who misarticulated only one sound, either /s/or/r/. Also, the subset of children who misarticulated /s/ was compared with children who misarticulated /r/ for performance on the measures classified above. Few of the clusters identified differed in articulation status or improvement with training.

Anthropometry↗