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Biomedical subjects

M Kearns

Publications and source records attributed to M Kearns.

At least 19 recordsLinked to original sources
PubMed2001

Spontaneous germline amplification and translocation of a transgene array.

The majority of the mammalian genome is thought to be relatively stable throughout and between generations. There are no developmentally programmed gene amplifications as seen in lower eukaryotes and prokaryotes, however a number of unscheduled gene amplifications have been documented. Apart from expansion of trinucleotide repeats and minisatellite DNA, which involve small DNA elements, other cases of gene or DNA amplifications in mammalian systems have been reported in tumor samples or permanent cell lines. The mechanisms underlying these amplifications remain unknown. Here, we report a spontaneous transgene amplification through the male germline which resulted in silencing of transgene expression. During routine screening one mouse, phenotypically negative for transgene expression, was found to have a transgene copy number much greater than that of the transgenic parent. Analysis of the transgene expansion revealed that the amplification in the new high copy transgenic line resulted in a copy number approximately 40-60 times the primary transgenic line copy number of 5-8 copies per haploid genome. Genetic breeding analysis suggested that this amplification was the result of insertion at only one integration site, that it was stable for at least two generations and that the site of insertion was different from the site at which the original 5-8 copy array had integrated. FISH analysis revealed that the new high copy array was on chromosome 7 F3/4 whereas the original low copy transgene array had been localised to chromosome 3E3. DNA methylation analysis revealed that the high copy transgene array was heavily methylated. The amplification of transgenes, although a rare event, may give insight into amplification of endogenous genes which can be associated with human disease.

Animals↗
PubMed2000

Complex patterns of inheritance of an imprinted murine transgene suggest incomplete germline erasure.

Here we report a transgenic mouse line that exhibits significant deviations from a classic pattern of parental imprinting. When the transgene is passed through the female germline, it is completely silenced in some offspring while in others expression is reduced. This variable expressivity does not appear to be the result of differences in the presence of unlinked modifiers. Female transmission of the transgene is associated with hypermethylation. The transgene is generally reactivated on passage through the male germline. Extended pedigrees reveal complex patterns of inheritance of the phenotype. The most likely explanation for this result is that the imprint is not completely erased and reset when passed through the germline of either sex. FISH analysis reveals that the transgene has integrated into chromosome 3 band E3, a region not known to carry imprinted genes, and the integration site shows no sign of allele-specific differential methylation. These findings, in conjunction with other recent work, raise the possibility that the introduction of foreign DNA into the mammalian genome, either through retrotransposition or transgenesis, may be associated with parental imprinting that is not always erased and reset during meiosis.

Animals↗
PubMed2000

Reactivation of heritably silenced gene expression in mice.

Epigenetic modifications that suppress gene activity in mammals are generally considered to be cleared in the germline, restoring totipotency of the genome. Here we report the germline inheritance of transcriptional silencing in mice, and reversion to activity after as many as three generations in the silent state. In a series of lines made with a LacZ transgene, one line exhibits variable expressivity: genotypically identical littermates have proportions of beta-Gal-positive erythrocytes that vary over at least four orders of magnitude, and in some offspring expression is completely silenced. The silent state of the transgene is inherited for multiple generations in the founder strain irrespective of the sex of the parent, implying maintenance of the epigenetic state through meiosis. Crosses of silenced mice with C57BL/6 mice result in reactivation of the transgene in approximately a third of F(1) littermates. The silencing involves a stochastic, all-or-none mechanism. Furthermore, silencing is transcriptional and correlates with methylation of the transgene as well as an inaccessible chromatin structure; these changes are reversed when expression is reactivated. This work supports the notion that silent genetic information in mammals can be inherited and later reactivated, and implies a mode of phenotypic inheritance that is less stable than Mendelian inheritance.

Animals↗
PubMed1999

Algorithmic stability and sanity-check bounds for leave-one-out cross-validation.

In this article we prove sanity-check bounds for the error of the leave-one-out cross-validation estimate of the generalization error: that is, bounds showing that the worst-case error of this estimate is not much worse than that of the training error estimate. The name sanity check refers to the fact that although we often expect the leave-one-out estimate to perform considerably better than the training error estimate, we are here only seeking assurance that its performance will not be considerably worse. Perhaps surprisingly, such assurance has been given only for limited cases in the prior literature on cross-validation. Any nontrivial bound on the error of leave-one-out must rely on some notion of algorithmic stability. Previous bounds relied on the rather strong notion of hypothesis stability, whose application was primarily limited to nearest-neighbor and other local algorithms. Here we introduce the new and weaker notion of error stability and apply it to obtain sanity-check bounds for leave-one-out for other classes of learning algorithms, including training error minimization procedures and Bayesian algorithms. We also provide lower bounds demonstrating the necessity of some form of error stability for proving bounds on the error of the leave-one-out estimate, and the fact that for training error minimization algorithms, in the worst case such bounds must still depend on the Vapnik-Chervonenkis dimension of the hypothesis class.

Algorithms↗
PubMed1996

Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.

Alleles↗
PubMed1996

Secretor status and human leucocyte antigens in coeliac disease.

BACKGROUND: The ability to secrete blood group antigens into body fluids and secretions is controlled by a single gene on chromosome 19. By means of erythrocyte Lewis (Le) antigen phenotype secretor status can be inferred. An increase prevalence of non-secretors of blood group antigens among coeliac patients has recently been described. METHODS: Blood was collected from 112 coeliac patients and 103 controls and tested for secretor status. Secretor status was correlated with human leucocyte antigens (HLA) in coeliac patients, thus evaluating a proposed interaction of susceptibility genes--that is, the secretor gene on chromosome 19 and HLA-linked genes on chromosome 6. Case notes for coeliacs were reviewed with regard to clinical outcome. RESULTS: Of 112 coeliacs who had either Le(a) or Le(b) antigens, 36 (32%) were non-secretors Le(a+, b-), compared with 27% (28) of 103 disease-free controls (P = 0.313). Recessive Lewis phenotype Le(a-, b-) was found in 9% of coeliacs versus 2% of controls. Prevalence of HLA-A1, B8, DR3, and DQ2 was unrelated to secretor status in coeliac versus patients. An increased prevalence of complications and coeliac-associated abnormalities was found in the non-secreting and recessive coeliac groups. CONCLUSIONS: This study shows no firm relationship between the non-secretor state and coeliac disease, nor any difference in the distribution of HLA markers among secretor and non-secretor coeliacs. It is unlikely, therefore, that the secretor gene is the much sought-after second coeliac gene.

Celiac Disease↗
PubMed1995

Position-dependent variegation of globin transgene expression in mice.

Expression of genes in eukaryotes has commonly been analyzed in a whole tissue, and levels of expression have been interpreted as the result of equivalent rates of transcription in every cell. We have produced transgenic mouse lines that express beta-galactosidase under the control of globin promoters linked to the major tissue-specific regulatory element of the alpha-globin locus, which permits the analysis of transgene expression in individual red blood cells. We find that expression of the transgene within all mouse lines is heterocellular. Individual cells either do not express the transgene at all or express it at a level characteristic of that line. The number of beta-galactosidase-expressing cells varies greatly between different lines of transgenic mice at any defined stage of development, but within a transgenic line, individual mice have strikingly similar numbers of expressing cells. This suggests that the degree of heterocellular expression is determined by the site of integration, as is seen in position-effect variegation.

Animals↗
PubMed1995

Results of a traditional acupuncture intervention for stuttering.

It is important that researchers investigate alternative strategies for treating stuttering, as contemporary treatments are not entirely successful in reducing stuttering with all people. Furthermore, many who have been "successfully" treated suffer from high relapse rates in the long term. Acupuncture has been shown to be a promising treatment for several diverse disorders, so a pilot investigation into its effectiveness for stuttering was considered worthwhile. This study investigated traditional acupuncture-based treatments for two adult males who had stuttered since childhood. A single-case experimental ABAB multiple baseline design was employed to test for treatment effectiveness. Subjects were followed up for a further 12 weeks to evaluate maintenance (C phase) of possible improvements. No significant ABAB reversal effects were observed, and stuttering frequency through the treatment phases remained at baseline levels for the two subjects. Speech rate also remained at baseline levels throughout the treatment phases, as did naturalness of speech and anxiety levels. This research is important as claims that acupuncture may successfully reduce stuttering need to be tested, and the scope and usefulness of treatments like acupuncture for a wide variety of problems needs to be determined. However, the low subject numbers involved suggest caution in concluding acupuncture is not a successful intervention for stuttering. Perhaps alternative acupuncture points need to be evaluated and a wider variety of persons who stutter need to be involved in any future research.

Acupuncture Therapy↗
PubMed1993

PAC-1: a mitogen-induced nuclear protein tyrosine phosphatase.

Tyrosine phosphorylation of proteins is required for signal transduction in cells and for growth regulation. A mitogen-induced gene (PAC-1) has been cloned from human T cells and encodes a 32-kilodalton protein that contains a sequence that defines the enzymatic site of known protein phosphotyrosine phosphatases (PTPases). Other than this sequence, PAC-1 is different from several other known related PTPases exemplified by PTP-1b. PAC-1 is similar to a phosphatase induced by mitogens or heat shock in fibroblasts, a yeast gene, and a vaccinia virus-encoded serine-tyrosine phosphatase (VH1). PAC-1 was predominantly expressed in hematopoietic tissues and localized to the nucleus in transfected COS-7 cells and in mitogen-stimulated T cells.

Amino Acid Sequence↗
PubMed1993

Treatment of choroidal neovascularisation in age-related macular degeneration with interferon alfa-2a and alfa-2b.

Forty eight eyes of 42 patients with choroidal neovascular membranes and age-related macular degeneration who received three different dose regimens of systemic interferon alfa-2 were studied retrospectively. The response to treatment of 41 eyes of the 37 patients who received at least 4 weeks' treatment was analysed with respect to the change in size of the choroidal neovascular membrane and the visual acuity compared with pretreatment levels. The size of the membrane at the end of the course of treatment had decreased in seven (17%) eyes overall, not changed in 16 (39%), and increased in 18 (44%). At the end of treatment, the visual acuity had improved in seven (17%) eyes, not changed in 27 (66%), and deteriorated in seven (17%). With an average follow up of 10 months after treatment, the visual acuity had deteriorated compared with the pretreatment value in 21 out of 41 (51%) eyes. Vision improved in some fellow eyes with disciform scars. Side effects were common and often severe. The data suggest that one of the major effects of interferon alfa may be to decrease vascular permeability. While further research may identify a place for interferon alfa in the treatment of choroidal neovascularisation, we were unable to demonstrate that the treatment regimens of systemic interferon alfa we used caused a dramatic benefit to patients with exudative age-related macular degeneration.

Aged↗
PubMed1985

Characterization of hematogenous cellular constituents of the murine decidua: a surface marker study.

Decidual tissue, which includes typical (stromal type) decidual cells as well as infiltrating leukocytes, appears to play a local immunoregulatory role in the maintenance of pregnancy in nature. The present study evaluated the contribution of numerous leukocyte subsets characterized on the basis of morphology combined with cell surface markers to the development of murine decidua during syngeneic (CBA female X CBA male) and allogeneic (CBA female X C57BL/6 male) pregnancy. Collagenase dispersed decidua were subjected to total and differential counts and cell surface labeling for a radioautographic identification of various markers: S-IgM on B cells, Thy-1 on T cells, neither marker on null lymphocytes, Lyt- (1 or 2 or 1,2) antigens on T cell subsets, Mac-1 and I-A on macrophages, using 125I-labeled monoclonal antibodies or a sandwich labeling with 125I-protein A. The total cellularity of decidua basalis showed a biphasic rise in both pregnancies, with peaks on day 11 and days 15 and 16, but the allopregnant decidua showed a higher accumulation of all cell types indicating that an allogeneic conceptus causes an augmented deciduogenesis. The number of decidual cells, the most frequent cell class, rose to a peak on day 11 followed by a decline possibly due to cell death. The number of lymphocytes, the next frequent cell class, showed a parallel pattern initially, followed by a sharp secondary rise on day 16. This rise may be due to a withdrawal of progesterone, an antiinflammatory hormone. Null cells predominated amongst decidual lymphocytes (45-80%), as well as in the progestational endometrium (53%), indicating a hormonal control of their accumulation. The frequency of B cells was low (10-13%) and T cells (25-45%) comparable to that in the blood, with Lyt-1 only class being the most common T cell subset. Allopregnant decidua also showed a late rise in the total number of Lyt-2 only cells which may have a suppressor function. Macrophages, the next common leukocyte class, all expressed Mac-1. Their number rose to a plateau by day 12, but at a higher level in allopregnancy. I-A (needed for antigen presentation) was expressed by an increasing proportion (5-60%) of macrophages with advancing gestation. These findings provide a basis for further functional studies.

Animals↗
PubMed1985

Radioautographic analysis of surface markers on decidual cells shared by cells of the lymphomyeloid tissues.

Stromal type decidual cells recovered from the murine decidua by a mild collagenase dispersion procedure contain immunoregulatory cells whose ultimate precursors may originate from the bone marrow. To explore the familial relationship of these cells with other cells of the immune system, a battery of cell surface markers recognized on lymphomyeloid cells were examined and quantitated at the morphological level on typical stromal type decidual cells of the dispersed CBA mouse decidua at 8-14 days of syngeneic pregnancy, using a sensitive radioautographic technique. Cells were either labeled directly by exposure to 125I-labeled monoclonal antibodies against Thy-1, Mac-1, or Lyt antigens or indirectly by a sequential exposure to monoclonal anti-I-Ak (Ia.17) or monospecific anti-I-JK antibodies and 125I-labeled Protein A. Decidual cells were found to be Thy-1+/- (13-73% positive, the incidence rising with advancing gestation in the decidua basalis), Mac-1+/- (present of 6-11% on day 8 and 17-32% on day 12), I-A-, 1-J-, and Lyt-. Macrophages within the decidua were Thy-1-, Lyt-, Mac-1+, and I-A+/- (present on 5-61% of cells, the incidence rising with advancing gestation).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗