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Biomedical subjects

J C Lewis

Publications and source records attributed to J C Lewis.

191 records · Page 11Linked to original sources
PubMedDate not supplied

Photoproteins as luminescent labels in binding assays.

Certain marine organisms produce calcium-activated photoproteins that allow them to emit light for a variety of purposes, such as defense, feeding, breeding, etc. Even though there are many bioluminescent organisms in nature, only a few photoproteins have been isolated and characterized. The mechanism of emission of light in the blue region is the result of an internal chemical reaction. Because there is no need for excitation through external irradiation for the emission of bioluminescence, the signal produced has virtually no background. This allows for the detection of the proteins at extremely low levels, making these photoproteins attractive labels for analytical applications. In that regard, the use of certain photoproteins, namely, aequorin, obelin, and the green fluorescent protein as labels in the design and development of binding assays for biomolecules has been reviewed. In addition, a related fluorescent photoprotein, the green fluorescent protein (GFP), has been recently employed in bioanalysis. The use of GFP in binding assays is also discussed in this review.

Amino Acid Sequence↗
PubMedDate not supplied

Observations on the treatment of necrobacillosis in wallabies.

Clinical experience of 73 cases of necrobacillosis in red-necked wallabies (Macropus rufogriseus) over a 6-year period is reviewed. Diagnostic methods and the medical and surgical treatment options are detailed. It is suggested that survival curves are necessary to assess the outcome of treatment.

Animals↗
PubMedDate not supplied

Microcomputer program for interactions in drug elimination in the rat.

In modern medicine patients often require multiple drug therapy. Such therapy can be modeled by a physiological flow model using interlinked differential equations to represent the differential rates of delivery and uptake of a drug by organs. Included in the model are the sites of action, toxicity, elimination and drug binding. A menu-driven computer program for this model was developed in FORTRAN 77 for execution on a microcomputer. The program computes the rate of uptake and concentration of drug in key tissues as a function of time. Drug administration can be modeled for bolus injection with up to 10 repeats and/or for continuous infusion of the drug. The drugs warfarin and adriamycin are used as illustrative examples. The program can handle multifactorial problems such as acute and chronic competitive elimination interactions, liver damage, and features of aging such as reduced drug binding and organ function. The computed results can be printed numerically or displayed graphically, either on a screen terminal or as hard copy. The results of several simulations may be cross-plotted for studies involving parametric changes such as would occur in multiple pathology and aging.

Animals↗
PubMedDate not supplied

C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development.

Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.

Aequorin↗
PubMedDate not supplied

Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization.

Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca(2+)-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO(2) and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.

Aequorin↗
PubMedDate not supplied

Site-specifically labeled photoprotein-thyroxine conjugates using aequorin mutants containing unique cysteine residues: applications for binding assays (Part II).

The jellyfish Aequorea victoria produces a protein, aequorin, which belongs to the class of Ca(2+)-dependent photoproteins known for their ability to emit visible light. This property of aequorin has allowed for its as a bioluminescent label in binding assays for a variety of analytes. Due to the excellent detection limits we demonstrated in assays for small peptides using a fusion protein between the peptide of interest and the photoprotein, our next goal was to expand the range of possible analytes for producing homogeneous populations of conjugates with the aequorin label to those that were nonpeptidic in nature. Recently, we prepared and characterized four aequorin mutants containing unique cysteine residues at various positions in the polypeptide chain. In the work reported here, the four aequorin mutants were each conjugated with a maleimide-activated methyl ester derivative of thyroxine, a hormone frequently determined to evaluate thyroid function. The thyroxine-aequorin mutant conjugates were characterized in terms of the bioluminescence activities and binding properties with an anti-thyroxine monoclonal antibody for possible future employment in either heterogeneous or homogeneous binding assays for thyroxine and/or other desired analytes.

Aequorin↗
PubMedDate not supplied

Normal haematological and serum biochemical values of Eurasian otters (Lutra lutra) from a Scottish rehabilitation centre.

Blood samples were taken from 47 clinically normal, wild-born Eurasian otters (Lutra lutra) before they were released from a rehabilitation centre in Scotland between August 1990 and March 1996. Serum biochemistry profiles were determined for 47 animals and haematological profiles for 41, and the results from males, females, and animals under or over one year of age were analysed as separate groups and as pooled populations. The normal ranges for a wide variety of haematological and serum biochemical parameters of the Eurasian otter are presented, and significant differences with age and sex are detailed.

Age Factors↗