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Biomedical subjects

C J Long

Publications and source records attributed to C J Long.

63 records · Page 4Linked to original sources
PubMedDate not supplied

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.

Acrylamides↗
PubMedDate not supplied

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.

Acrylamides↗
PubMedDate not supplied

Site-specific polymer-streptavidin bioconjugate for pH-controlled binding and triggered release of biotin.

Low molecular weight copolymers of acrylic acid (AAc) and N-isopropylacrylamide (NIPAAm) have been synthesized with reactive OH groups at one end, using a chain transfer polymerization technique. The copolymer displays both pH and temperature sensitivity over a wide and useful range of pHs and temperatures, which permits both pH and temperature control of polymer conformation. This copolymer has been conjugated to a specific cysteine thiol site inserted by genetic engineering near the recognition site of streptavidin (SAv). In this paper, we demonstrate that this bioconjugate can provide pH control of biotin binding to and triggered release from the mutant SAv. These actions are relevant to affinity separations, biosensors, diagnostics, enzyme processes, and targeted delivery of drugs or chemical agents, labels, and other signals.

Acrylamides↗
PubMedDate not supplied

Remediation of alexia without agraphia: a case study.

Following a left temporoparietal-occipital haemorrhage and surgery, a 43-year-old, right-handed male exhibited alexia without agraphia. A remediation programme consisted of training in head turning to compensate for a right visual field defect, letter-by-letter reading aloud and covertly, drill with flash-cards to improve word recognition and practice in naming objects to improve dysnomia. The patient's reading improved markedly over a 6-week period and he was able to resume work as a respiratory therapy supervisor. A post-morbid depression resolved concomitantly with the patient's return to work. The training programme and the patient's post-training approach to reading are discussed in terms of hemispheric functioning as well as 'direct path' and 'indirect path' reading. The effectiveness of training is considered in the context of spontaneous recovery.

Adult↗
PubMedDate not supplied

Depression among the head-injured and non-head-injured: a discriminant analysis.

Neuropsychologists often use traditional psychological tests to assess depression following a head injury; but the assumption that depression with a head injury resembles that in an uninjured person is suspect. The current study attempts to examine the cognitive manifestations of depression with and without a coexisting head-injury. Advanced statistical methods are used to assess whether or not the two depressions 'look alike' with respect to the neuropsychological sequelae of the disorders. A total of 1182 people were entered into one of two discriminant function analyses (DFA) for depression. Each person was a member of one of the following groups: (a) depressed, (b) non-depressed, (c) head-injured, or (d) head-injured and depressed. Two functions were performed for depression, one on the population of head-injured people and one on the population of uninjured people. Cross-validations were performed for each population and across populations in order to assess the utility of each population's function for the opposite group. This comparison allows the researcher to indirectly compare depression in the two populations. Both functions were successfully applied to either population when MMPI variables were included in the analyses. However, when only cognitive variables were included the function performed on the non-head-injured population did not correctly classify head-injured people as depressed or non-depressed. One explanation for this is that the range of cognitive scores in head-injured people is so great that it allows for a less accurate but more generalizable function. Suggestions for future research are discussed.

Adult↗