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Biomedical subjects

D Stephenson

Publications and source records attributed to D Stephenson.

16 recordsLinked to original sources
PubMed1996

Nuclear and cytoplasmic localization of the beta-amyloid peptide (1-43) in transfected 293 cells.

Cultures of transformed human embryonic kidney 293 cells were transiently transfected with minigene constructs coding for the Abeta peptide (1-43). The Abeta minigene used in this study consisted of exons 16 and 17 of the amyloid precursor protein gene, including the 6000+ bp intronic region. Two of the constructs used in this study, human amyloid precursor protein (APP) promoter-driven Abeta minigene and BK virus enhancer/adenovirus major late promoter-driven Abeta minigene, did not contain a signal peptide sequence, whereas the third, human APP promoter-signal peptide Abeta minigene did not contain the human APP signal sequence. The resulting Abeta products were detected by immune precipitation, using 10D5 antibody and Western blot analysis, using R1280 antisera, as SDS stable oligomers in cell lysates of cells containing all three constructs or in culture media when produced by the signal peptide construct. Evaluation of the cells by immunocytochemistry using conventional and transmission electron microscopy indicated that the cells transfected with constructs without the signal peptide accumulated immunoreactive Abeta primarily in the nucleus.

Amyloid beta-Peptides↗
PubMed1996

Induction of sulfated glycoprotein-2 (clusterin) and glial fibrillary acidic protein (GFAP) RNA expression following transient global ischemia is differentially attenuated by LY231617.

Sulfated glycoprotein-2 (SGP-2) is a secreted glycoprotein that along with GFAP has emerged as a prominent molecular marker of neurodegeneration. In the present study, we have evaluated further the relationship between SGP-2, GFAP and neurodegeneration, by examining the effects of LY231617, a potent antioxidant, on expression of SGP-2 and GFAP following four vessel occlusion (4VO). GFAP and SGP-2 RNA levels increased several fold in hippocampus and caudate nucleus in response to 30 min of 4VO. LY231617 treatment markedly attenuated the induction of GFAP RNA in both hippocampus and caudate nucleus, consistent with the significant neuroprotection observed histologically. In contrast, LY231617 treatment blunted SGP-2 RNA expression only in the hippocampus; SGP-2 RNA expression in caudate nucleus was similar to vehicle-treated 4VO, despite the marked attenuation of neuronal damage in both areas by LY231617. These data suggest region-specific differential regulation of SGP-2 and GFAP RNA induction.

Animals↗
PubMed1995

Molecular markers near two mouse chromosome 13 genes, muted and pearl, which cause platelet storage pool deficiency (SPD).

The recessive muted (mu) and pearl (pe) mutations on Chromosome (Chr) 13 cause pigment dilution and platelet storage pool deficiency (SPD) in mice. In addition, mu causes inner ear abnormalities and pe has symptoms associated with night blindness. Using an interspecific backcross involving the wild-derived Mus musculus musculus (PWK) stock, we have mapped 33 microsatellite markers and four cDNAs relative to mu, pe, and another recessive mutation, satin (sa). Analyzing a total of 528 backcross offspring, we found tight linkage between the pigment loci and several microsatellite markers (D13Mit87, D13Mit88, D13Mit137 with mu; and D13Mit104, D13Mit160, D13Mit161, and D13Mit169 with pe). These markers should aid the eventual molecular identification of these specific SPD genes.

Animals↗
PubMed1993

Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal.

Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP-binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP-binding proteins may affect one or both processes.

Animals↗
PubMed1993

Decrease in amyloid precursor protein precedes hippocampal degeneration in rat brain following transient global ischemia.

We have studied amyloid precursor protein (APP) expression in rat brain following transient global ischemia. Ischemic damage 24 h after 30 min of four-vessel occlusion (4VO) was limited to the caudate nucleus; hippocampal pyramidal neurons appeared histologically normal by light microscopy. Consistent with ongoing neurodegeneration in the caudate nucleus, microtubule-associated protein-2 (MAP-2) levels assessed by immunoblots were significantly reduced in homogenates of caudate nucleus after 4VO. MAP-2 levels In the hippocampus were comparable to control values. In contrast, full length APP levels in both the caudate nucleus and hippocampal homogenates were significantly decreased following 4VO despite normal hippocampal morphology at 24 h. These findings suggest that decrements in full length APP precede overt neuronal damage and may play a role in the subsequent delayed neurodegeneration in the hippocampus.

Amyloid beta-Protein Precursor↗
PubMed1993

Close physical linkage of the FLT1 and FLT3 genes on chromosome 13 in man and chromosome 5 in mouse.

Receptor-type tyrosine kinases (RTK) with five or seven immunoglobulin-like domains in their extracellular region are encoded by genes grouped in clusters. In human, two such clusters have been individualized, in chromosomal regions 4q11-q12 and 5q33-qter respectively. We define here a third cluster located on chromosome 13q and containing two contiguous RTK genes, FLT1 and FLT3. The former has recently been shown to encode a RTK of a new class while the latter codes for a hematopoietic receptor closely related to the products of the FMS and KIT genes. The physical linkage is also evidenced in mouse, where the two genes appear to lie within a 350 kb Mlu I fragment, on mouse chromosome 5.

Animals↗
PubMed1992

Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia.

Sulfated glycoprotein-2 (SGP-2) is emerging as a prominent marker of neurodegeneration in mammalian brain. Regulation of brain SGP-2 was studied in adult male Wistar rats subjected to 30 min of forebrain ischemia by four vessel occlusion. By 3 days after the ischemic insult, SGP-2 RNA levels were increased two fold in caudate nucleus and hippocampus. SGP-2 protein levels assessed by immunoblots were markedly increased in both brain regions following ischemia. GFAP RNA levels also increased over 5 fold in caudate nucleus and hippocampus following the ischemic insult. Despite significant elevations in GFAP RNA, protein levels of GFAP assessed by immunoblot were only marginally affected. The elevated expression of SGP-2 in rodent brain following this and other experimental lesion paradigms (e.g., excitotoxic lesions, deafferentation) suggest some general involvement of SGP-2 in neurodegeneration and remodelling following neuronal injury.

Animals↗
PubMed1985

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm.

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.

Animals↗
PubMed1983

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry.

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.

Animals↗
PubMed1982

Sex preselection in mammals? Separation of sperm bearing Y and "O" chromosomes in the vole Microtus oregoni.

The two sex determining sperm populations of the vole Microtus oregoni were separated according to DNA content by use of flow sorting instrumentation. Although the sperm were not viable, they should be useful for addressing the question of haploid expression of genes linked to sex chromosomes and for efficiently searching for biochemical markers that differentiate the two populations.

Animals↗
PubMed1982

High resolution DNA content measurements of mammalian sperm.

The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +/- 7 degrees. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to X] translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

Acriflavine↗
PubMed1974

Pseudomembranous colitis associated with antibiotic therapy - an emerging entity.

Two cases of pseudomembranous colitis are presented. The first patient had been treated with novobiocin-tetracycline and penicillin, and two weeks later developed severe fulminating diarrhea with ascites and bilateral pleural effusions which did not respond to intravenous ACTH. Subsequently she underwent subtotal colectomy and made a rapid and complete recovery. The second patient developed severe diarrhea two weeks after a 10-day course of clindamycin. She was treated with intravenous ACTH, oral Lactobacillus and a fecal enema and made a complete recovery.These cases reconfirm the importance of antibiotics as etiologic agents in this disease. They also stress the classic sigmoidoscopic and histologic findings that should facilitate prompt and rapid diagnosis.

Adolescent↗
PubMedDate not supplied

Shear thickening of DNA in SDS lysates.

Cells of Escherichia coli were subjected to lysis by an alkali detergent treatment (sodium dodecyl sulphate). The rate of detergent lysis was not first order. The rheological properties of the lysate were measured using a controlled stress rheometer. Detergent-lysed cells produced a lysate which showed marked non-Newtonian properties. The material showed a shear thickening at specific low shear stress conditions. The acceleration of the cone during the ascent stage of a flow curve, influenced the shape of the flow curves. These findings are discussed in relation to the form of bacterial DNA in solution.

DNA, Bacterial↗