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Biomedical subjects

J L Hopkins

Publications and source records attributed to J L Hopkins.

15 recordsLinked to original sources
PubMed2001

Binding site elucidation of hydantoin-based antagonists of LFA-1 using multidisciplinary technologies: evidence for the allosteric inhibition of a protein--protein interaction.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

Allosteric Site↗
PubMed1994

High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus.

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Affinity Labels↗
PubMed1993

Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase.

The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis.

Amino Acid Sequence↗
PubMed1991

Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.

3C Viral Proteases↗
PubMed1982

Detection and recognition masking-level differences for the individual CID W-1 spondaic words.

The detection and recognition masking-level differences for the 36 individual CID W-1 spondaic words (Technisonic Studios) were established on 36 listeners with normal hearing. The masking for two conditions, SoNo and S pi No, was accomplished with 70-dB-SPL speech-spectrum noise. The word lists were administered in 2-dB decrements with the 36 words presented at each signal-to-noise ratio, which ranged from 9 dB to -19 dB for SoNo and from 3 dB to -25 dB for S pi No. The detection and recognition thresholds determined for each spondaic word under both masking conditions are presented. Additionally, the slopes of the psychometric functions for each word under both conditions are given. The mean masking-level difference (SoNo-S pi No) for the detection-response task was 9.4 dB (SD = .7 dB). The mean masking-level difference for the recognition task was 7.2 dB (SD = 1.4 dB). The recognition masking-level differences for the individual spondaic words ranged from 4.4 dB (stairway) to 10.0 dB (oatmeal). A list of 10 words that had the largest masking-level differences for recognition is suggested to enhance the clinical utility of the masking-level difference for speech.

Auditory Threshold↗
PubMed1979

Chalcosis: a study of natural history.

Ten patients harboring a small, intra-vitreal, copper foreign body in one eye for 4 months to 29 years have been studied clinically and electrophysiologically. Nine of the ten affected eyes had a visual acuity of 20/60 or better. Some asymmetry existed in the electrophysiologic responses of seven affected eyes as compared to the controls, but all of these responses were within the normal range. The electroretinogram was reduced in one eye but was consistent with vitreous opacification. Vitreous opacification and copper deposition in the macula were potentially serious sequelae observed in four eyes. Small, intraocular copper foreign bodies can be tolerated for long periods of time without retinal toxicity, but vitreous changes of maculopathy may necessitate intervention.

Adolescent↗
PubMed1978

Use of platelet-fibrinogen-thrombin mixture to seal experimental penetrating corneal wounds.

A platelet-fibrinogen-thrombin mixture utilizing autologous platelets was studied for its potential to seal perforating corneal wounds. In rabbits, the mixture demonstrated sufficient adhesive properties to permit 75% of penetrating keratoplasties to remain in place without the use of sutures. All 12 grafts held initially; after 48 h three grafts extruded. In control eyes only two out of 12 (16%) of the corneal grafts remained in place. The material is simple to prepare and apply and is not toxic to the cornea. It does not cause inflammation or lid irritation. Intraocular complications observed with its use were transient anterior synechiae and retrocorneal membrane formation.

Animals↗
PubMed1978

Mass visual screening via television.

Videotaped programs that could be used for screening of visual acuity and of visual fields by means of broadcast television have been developed and tested. The tests are designed to be effective regardless of the size of the viewer's television screen. Inevitably, false-positive and false-negative results would occur, which would have to be carefully explained at the time the tests were broadcast.

Adolescent↗
PubMed1978

Chloroquine retinopathy in the rhesus monkey.

Chloroquine was administered intramuscularly 5 days a week to rhesus monkeys for as long as 4 1/2 years. No clinical, fluorescein angiographic, or electrophysiological evidence of retinal damage was observed. Yet chloroquine/chloroquine byproduct analysis of the ocular tissues revealed an enormous binding capacity of the pigmented tissues of the eye (choroid plus RPE, ciliary body, and iris) with eventual accumulation observed in the retina. Despite the normal ophthalmic appearance and function, extensive pathological changes occurred in the retinas and choroids of these experimental monkeys. The chloroquine caused an initial dramatic effect on the ganglion cells, with the photoreceptors affected shortly thereafter. Patching degeneration of the ganglion cells and photoreceptors then progressed over several years, with the choroid and pigment epithelium ultimately deteriorating as well.

Animals↗
PubMed1976

Intraocular copper foreign bodies. Use of dexamethasone to suppress inflammation.

In an attempt to prevent the intense inflammatory response to intravitreal copper foreign bodies in the rabbit eye, varying dosages of dexamethasone sodium phosphate were administered daily via the subconjunctival route. The steroid appeared to behave systemically and had similar effects in both the injected and the noninjected eyes. Its effect was similar also regardless of the concentration of copper within the foreign body. Nine days of dexamethasone therapy prevented a substantial number of copper foreign bodies from becoming totally encapsulated during that time. Furthermore, a delay in the onset of the inflammatory response to the foreign body was noted. In a few instances, inflammation was completely suppressed during the entire course of therapy.

Alloys↗