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Biomedical subjects

N C Nelson

Publications and source records attributed to N C Nelson.

At least 37 records · Page 2Linked to original sources
PubMed1991

A technique for retinal pigment epithelium transplantation for age-related macular degeneration secondary to extensive subfoveal scarring.

We describe the surgical excision of submacular scar in end-stage age-related macular degeneration and transplantation of autologous and homologous retinal pigment epithelial (RPE) cells. The technique involves the preparation of a large retinal flap encompassing the macula and the arcades, removal of the submacular scar, and replacement of the RPE cells, using either an autologous pedicle graft or homologous RPE cells and Bruch's membrane. Fourteen months following the procedure, visual acuity in a patient with a pedicle graft had improved from counts fingers to 20/400 and the patient fixated over the transplanted RPE cells. After 10 months, a homologous graft in a second patient had become encapsulated with a fine subretinal membrane without neovascular tissue; visual acuity had not improved. No intraoperative or postoperative complications resulting from the surgery occurred in either patient.

Aged↗
PubMed1990

Chemiluminescent DNA probes: a comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays.

Nucleic acid hybridization has the potential to markedly improve the diagnosis of infectious and genetic diseases. Recently, chemiluminescent hybridization assays using acridinium esters and stabilized dioxetanes have been described with sensitivities comparable to those obtained with radioactive labels. Acridinium esters are used as direct labels that are attached to the probe throughout the hybridization reaction. Methods have been developed for labeling DNA probes with acridinium esters at high specific activity and for stabilizing the label under the relatively harsh conditions of hybridization reactions. The label does not affect the kinetics of the hybridization reaction or the stability of the resulting hybrid. The label emits light upon exposure to alkaline peroxide; thus, the assay format can be an extremely simple one. The acridinium ester labels are stable in storage and exhibit extremely rapid light-off kinetics which permit reading large numbers of samples within a brief period as well as limiting the contribution of background signal. A special property of acridinium esters allows chemical destruction of the label when it is present on unhybridized probe, whereas the label is stable to this process when the probe is hybridized. This behavior forms the basis of techniques to minimize assay background signals and allows a homogeneous assay format which does not require physical separation of hybridized and unhybridized probe. The adamantyl-stabilized 1,2-dioxetanes have been used to produce high-sensitivity detection systems for clinical assays. The probe is labeled with enzymes such as alkaline phosphatase or beta-D-galactosidase that hydrolyze the dioxetane derivative to produce a chemiluminescent molecule. As with other enzyme-based labeling systems, the signal increases with time, allowing greater sensitivity to be achieved with longer incubations. The amount of light generated is sufficient to expose sensitive photographic film with extended incubation; therefore, convenient assay formats not requiring instrumentation can be used. Excellent analytical sensitivities have been reported, and by using labels with different light emissions and/or different enzymes on the probes, it is possible to distinguish multiple target sites within a single assay. Because the label is suited for use with solid supports such as polyacrylamide gels, membrane filters, or microscope slides, applications include DNA sequencing, dot and Southern blot hybridizations, and in situ hybridization.

Acridines↗
PubMed1989

Assay formats involving acridinium-ester-labeled DNA probes.

We describe the development of several hybridization assay formats involving acridinium-ester-labeled DNA probes. The simplest of these is a homogeneous assay procedure that requires only three steps to complete, including a 5-s detection step. Using this format, we have detected target sequences in the 10(-16) to 10(-17) mol range; when rRNA is the target, this translates to 3000 to 300 bacterial organisms. The entire assay can be carried out in less than 30 min. This is the first homogeneous DNA probe assay to be of practical use in the clinical laboratory, and it represents a major simplification of hybridization formats. We also demonstrate the use of this homogeneous assay format to discriminate single-base differences between two closely related target sequences and to detect DNA as well as RNA target molecules. By combining homogeneous hybrid discrimination with solid-phase separation, we have been able to decrease background readings from unhybridized probe to only a few parts per million. This enhances assay sensitivity about 10-fold, to a range of 10(-17) to 10(-18) mol of target. We are in the process of further improving the performance of these assays.

Acridines↗
PubMed1983

Selective protection of sulfhydryl groups in cAMP-dependent protein kinase II.

The cysteine residues of cAMP-dependent protein kinase II from porcine heart have been probed using alkylation with iodoacetic acid. Alkylation of the dissociated catalytic subunit resulted in loss of activity that was concomitant with the alkylation of both cysteine residues, Cys 199 and Cys 343. In contrast, no loss of activity was observed following alkylation of the holoenzyme. Isolation of the C-subunit following dissociation of the alkylated holoenzyme with cAMP revealed that Cys 343 was fully alkylated whereas Cys 199 was completely protected from chemical modification. These results establish that Cys 343 is not essential for enzymatic activity and, furthermore, indicate that aggregation of the C- and R-subunits selectively protects Cys 199. Alkylation of the R-subunit was also characterized both in the presence and absence of C-subunit. Cysteines 97, 124, and 326 were alkylated in R2 both in the presence and absence of cAMP. Of these 3 residues only Cys 97 was protected from alkylation in the holoenzyme. In the presence of high concentrations of iodoacetic acid, partial alkylation of the remaining 3 cysteine residues was observed, and this labeling was eliminated by cAMP suggesting that cAMP results in a general tightening of the protein conformation rather than the selective protection of any single cysteine residue.

Alkylation↗
PubMed1982

Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates.

The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198.

Affinity Labels↗
PubMed1982

A case-comparison study of hypertension and hyperparathyroidism.

The frequency of hypertension in patients with primary hyperparathyroidism has been compared with that found in cases matched for conditions of comparable surgical magnitude, age, race, sex, and days in the hospital. The mean blood pressure of the patients was 143/89 mm Hg, significantly higher than that of controls, which was 130/81 mm Hg (P less than 0.001 for systolic and diastolic pressures). The difference was especially notable in view of the fact that the mean weight of the controls was 169 lb compared to 153.9 lb for the patients. Actual hypertension, defined as a diastolic blood pressure of 90 mm Hg or more and or treatment with hypertensive therapy was much more frequent in the patients (90 of 124; 73%) than in the controls (53 of 124; 43%; P less than 0.001). Serum creatinine was 1.3 mm/dl in patients and 1.1 mm/dl in controls. The blood pressure difference between patients and controls persisted when patients with creatinine levels of 2.0 mm/dl or less were omitted. The blood pressure difference between patients and controls had disappeared by the end of hospitalization. We conclude that primary hyperparathyroidism is associated with modest blood pressure elevation, that renal damage is probably not the mechanism, and that surgical cure perhaps may lower blood pressure. The mean effect of primary hyperparathyroidism is to shift the blood pressure distribution curve to the right by approximately 15/10 mm Hg.

Age Factors↗
PubMed1981

Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71.

p-Fluorosulfonylbenzoyl 5'-adenosine (FSO2BzAdo) was shown previously to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase II from porcine skeletal muscle (Zoller, M. J., and Taylor, S. S. (1979) J. Biol. Chem. 254, 8363-8368). The catalytic subunit of porcine heart cAMP-dependent protein kinase was also inhibited following incubation with FSO2[14C]BzAdo, and inhibition was shown to result from the stoichiometric, covalent modification of a single lysine residue. The amino acid sequence in an extended region around the carboxybenzenesulfonyl lysine (CBS-lysine) was elucidated by characterizing both tryptic and cyanogen bromide peptides containing the 14C-modified residue. The sequence in this region was Leu-Val-Lys-His-Lys-Glu-Thr-Gly-Asn-His-Phe-Ala-Met-Lys(CBS)-Ile-Leu-Asp-Lys-Glu-Lys-Val-Val-Lys-Leu-Lys-Gln-Ile. The covalently modified residue corresponded to lysine 71 in the overall polypeptide chain. Homologies to bovine heart catalytic subunit and to a site modified by FSO2BzAdo in phosphofructokinase are considered.

Adenosine↗
PubMed1975

Microchromatography of hemoglobins. V. Thin-layer chromatography of some hemoglobins on CM-cellulose.

By means of thin-layer ion-exchange chromatography, human hemoglobins A, S, and C in various combinations can be distinctly separated. Also, hemoglobin F can be separated from the A, S, and C components of cord blood samples from infants and in adult blood samples with a relatively high percentage of F (15% or more). The procedure uses CM-cellulose and solutions of Tris-HCl, NaCl, and KCN. A 0.3-mg sample in 5-10 mul of solvent is required. Development can be completed in 30-40 min.

Age Factors↗