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Biomedical subjects

C R Merril

Publications and source records attributed to C R Merril.

At least 91 records · Page 5Linked to original sources
PubMed1991

Abnormal protein in the cerebrospinal fluid of patients with a submicroscopic X-chromosomal deletion associated with Norrie disease: preliminary report.

Norrie disease is an X-linked recessive disorder characterized by congenital blindness and, in many cases, mental retardation. Some Norrie disease cases have been shown to be associated with a submicroscopic deletion in chromosomal region Xp11.3. Cerebrospinal fluid (CSF) was collected from four male patients with an X-chromosomal deletion associated with Norrie disease. CSF proteins were resolved using two-dimensional gel electrophoresis and then analyzed by computer using the Elsie V program. Our analysis revealed a protein that appears to be altered in patients with Norrie disease deletion.

Blindness↗
PubMed1990

Noradrenergic control of the synthesis of two rat pineal proteins.

Pineal physiology is controlled by norepinephrine released from sympathetic nerves terminating in the gland. In the present study, the effect of norepinephrine on the labelling of specific proteins was investigated by incubating glands with [35S]methionine and then resolving the proteins by two-dimensional polyacrylamide gel electrophoresis; the patterns were analyzed by computer-assisted image analysis. The most prominent effects of norepinephrine were distinct and consistent increases in the labelling of two proteins (37 kDa, pI = 6.0, 50 kDa, pI = 6.0), designated adrenergically induced protein (AIP 37/6 and AIP 50/6). In both cases, norepinephrine was effective at low concentrations (EC50 = 10 nM). Pharmacological studies indicated that the effects of norepinephrine on both proteins involved a beta-adrenergic receptor, and that cyclic AMP was the second messenger. Pulse-chase labelling experiments revealed that these effects of norepinephrine did not involve post-translational modification of previously labelled precursor proteins, but depended upon de novo synthesis of protein. An inhibitor of mRNA synthesis, actinomycin-D, was found to block the effect of norepinephrine on AIP 50/6 but not on AIP 37/6, suggesting that norepinephrine acted on AIP 50/6 via a transcriptional mechanism and on AIP 37/6 via a translational mechanism. These in vitro studies were extended into in vivo investigations by measuring silver-stained AIP 37/6 in the two-dimensional gels. Changes in the amount of AIP 37/6 in pineal glands were studied in response to treatments which block the adrenergic stimulation of the gland, including exposure to constant lighting or removal of the superior cervical ganglia. Both treatments reduced AIP 37/6 by 50-75% in 8 weeks. These observations, together with those from in vitro studies, suggest that the amount of AIP 37/6 in the pineal gland is regulated by norepinephrine; and further, that norepinephrine acts through a beta-adrenergic-cyclic AMP mechanism to control AIP 37/6 synthesis at a translational level.

Adrenergic Fibers↗
PubMed1990

Silver staining of proteins and DNA.

Silver stains offer high sensitivity for the detection of proteins and DNA separated on gels and membranes. These stains depend on the reduction of ionic to metallic silver.

Ammonia↗
PubMed1990

Sodium pentosan polysulfate (PPS), an anti-HIV agent also exhibits synergism with AZT, lymphoproliferative activity, and virus enhancement.

Sodium pentosan polysulfate (PPS), a negatively charged polymer of beta-D-xylopyranose units, was evaluated for its anti-HIV effects in normal human peripheral mononuclear cells (PMNC) and its possible synergism with AZT. In the presence of 25 nM AZT, 2.0 micrograms/ml of PPS reduced HIV-1 replication 110-fold, compared with a 3.9- and 7-fold decrease in the presence of either drug individually. Surprisingly, at low (below 1 microgram/ml) concentrations of either PPS or dextran sulfate, an enhancement of virus production was observed. PPS was nontoxic, had a proliferative effect on uninfected and a protective effect on infected PMNC. Virus enhancement at low concentrations of PPS appeared to be linked to its lymphoproliferative effect. These findings suggest that the use of PPS and others such agents as monotherapy for AIDS might have deleterious effects. However, due to its marked synergism with AZT and its lymphoproliferative activities, PPS might prove to be a useful agent in therapeutic trials of AIDS if used in combination with less than the usual dosage of AZT.

Carbohydrate Sequence↗
PubMed1989

Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction.

A method to rapidly generate single stranded DNA for dideoxy sequencing following the polymerase chain reaction is described. By incorporating biotin in one of the amplification primers, we are able to physically separate the two DNA strands produced in the polymerase chain reaction. After amplification, the mixture is passed through a column containing streptavidin agarose. The strand produced by the biotinylated primer is bound in this matrix. The unbiotinylated strand is eluted with 0.2 N NaOH and sequenced by the dideoxy method. This method was utilized to sequence mitochondrial DNA from crude genomic DNA and to determine the sequences of four clones containing human mitochondrial DNA as a test of its accuracy. The use of biotin-facilitated separation permitted us to amplify and sequence DNA samples in a single day.

Autoradiography↗
PubMed1989

Release from live choroid plexus of apical fragments and electrophoretic characterization of their synthetic products.

Protein synthesis and secretion by the choroid plexus (CP) has been implicated as a major source of certain proteins in cerebrospinal fluid (CSF), such as transthyretin. The suggestion that proteins are elaborated from CP through apocrine secretion has been borne out by the presence of newly labeled proteins in apical protrusions from CP (Agnew et al.: Cell and Tissue Research 208:261-281, 1980a). When the protrusions (aposomes) separate from the cells, they continue to incorporate labeled amino acids (Gudeman et al.: Tissue and Cell 19:101-109, 1987). In the present work the formation of aposomes in live CP explants indicated that these spheroids were not the result of fixation. Aposomes were also identified within rat CSF by immunohistochemistry with monoclonal directed against aposomes as well as with anti-transthyretin serum. The protein product of aposomes was characterized by 2-dimensional SDS-PAGE and compared to the protein products of whole CP tissue. Paradoxically, transthyretin, a heavily labeled protein in the tissue, was virtually undetected in the aposome synthetic profile. However, four other proteins were expressed in relatively equivalent amounts by the aposomes. The presence of mRNA in aposomes was detected with a poly dT probe, and the presence of actin was revealed by phalloidin staining of aposomes. These studies provide a more comprehensive definition of aposomes, but the functions of their secreted proteins remains to be determined.

Animals↗
PubMed1989

Use of image analysis to quantitate changes in form of mitochondrial DNA after x-irradiation.

In order to develop a sensitive method that can reliably assess the amount of previous genotoxic exposure, we examined whether relative loss of the supercoiled form of mitochondrial DNA in tissue culture cells could retrospectively reflect exposure to x-irradiation of 375,750, and 1500 roentgens. Five routines of a graphic analysis program named IMAGE developed for the Apple Macintosh II computer were used to densitometrically quantitate the relative amounts of supercoiled and nicked-circular forms of mitochondrial DNA blotted onto nitrocellulose. After x-irradiation, there were dose-dependent losses of supercoiled relative to nicked-circular DNA forms. Further developments in the methodology to evaluate changes in form of mitochondrial DNA should increase the sensitivity and reliability of the dosimetry. The IMAGE program is useful for a wide variety of studies in molecular biology, and is available at no charge.

Animals↗
PubMed1988

Cerebrospinal fluid protein analysis in diseases of the nervous system.

Electrophoretic studies of human CSF proteins from patients with diseases of the NS are reviewed. Various 1-DE methods are of similar value in identifying the non-specific OBs, which are helpful in the diagnosis of MS and recurrent GBS. In early and subclinical MS, OBs are of prognostic value, with IEF gels having the greater resolution. Silver-stained 2-DE gels provide the equivalent information to the OBs on 1-DE gels, with even greater sensitivity, and yield additional disease-associated protein data. Two proteins have proven to have diagnostic value in CJD and other changes that are still being evaluated have been identified in Parkinson's disease, GBS, Alzheimer's disease, schizophrenia and Herpes simplex encephalitis. The vastly improved CSF protein information obtained with silver-stained 2-DE gels heralds both a change from the relatively limited applications with 1-DE methods and also the need to adopt this approach in the routine clinical laboratory.

Animals↗
PubMed1988

Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining.

Background staining that is associated with silver detection of proteins and nucleic acids in polyacrylamide gels has been shown to be due mostly to the amide groups in methylenebisacrylamide, a commonly used gel crosslinker. In attempts to reduce this background staining, eight existing crosslinking agents were tested. All of these proved to be unsuitable. Six new crosslinking agents were synthesized and tested. Of these, diacrylylpiperazine provided increased physical strength, improved electrophoretic separation of proteins, and silver staining detection of proteins with reduced background stain.

Acrylamides↗
PubMed1988

Methods for increasing the resolution of two-dimensional protein electrophoresis.

A two-dimensional gel elctrophoresis protocol has been developed which provides for a 1.5-to 3-fold increase in the resolution of proteins compared to other frequently used methods. The major variations from previous protocols include increased pore size in the isoelectric focusing gels; cholamidopropyldimethylhydroxypropanesulfonate, a zwitterionic detergent, replaces most of the Nonidet P-40, a nonionic detergent, in the isoelectric focusing gels; no equilibration step is employed between the first and second dimensional separation. The use of a stacking gel in the second dimension has been eliminated; a more efficient and evenly distributed cooling system has been designed for the molecular mass separation, allowing faster migration with higher current. Finally, the crosslinker diacrylylpiperazine is employed which improves protein separation and detection with ammoniacal silver staining. Silver-stained two-dimensional gel electrophoretograms of human plasma and hamster brain tissues and autoradiographs of rat liver cells are compared to the results obtained from previous methods.

Animals↗
PubMed1988

Coloration of silver-stained protein bands in polyacrylamide gels is caused by light scattering from silver grains of characteristic sizes.

This study investigates the physical basis of color effects in the detection of proteins in polyacrylamide gels by silver staining. Specifically, the hypothesis that different colors may correlate with the development of silver grains of characteristic sizes was investigated by electron microscopy. Protein bands that stained brown, yellow, and blue were excised from stained gels and prepared for electron microscopy by thin-sectioning. In each case, the size distributions of globular silver grains were determined directly from the electron micrographs. We found that blue bands have larger silver grains (with diameters of 40-100 nm) than yellow (21-39 nm) or brown bands (17-35 nm). On the basis of these and other observations, a general mechanism is proposed whereby chemical specificity of electrophoretically separated proteins is expressed in color-specific silver staining.

Electrophoresis, Polyacrylamide Gel↗