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L Bowden

Publications and source records attributed to L Bowden.

At least 19 recordsLinked to original sources
PubMed2001

An intragenic methylated region in the imprinted Igf2 gene augments transcription.

DNA methylation is usually associated with transcriptional silencing, but in the imprinted mouse Igf2 gene, the paternally expressed copy is methylated in two discrete differentially methylated regions (DMRs). DMR1 is located upstream of the fetal promoters and has been shown to be a methylation sensitive silencer. Here we examine the role of the intragenic DMR2 by gene targeting. In contrast to DMR1, deletion of DMR2 on the maternal allele did not lead to activation of the silent Igf2 gene. Deletion of a 54 bp methylated core region in DMR2 on the paternal allele, however, reduced Igf2 mRNA levels and was associated with fetal growth retardation. Nuclear run-on assays showed that the core region influenced transcription initiation, and luciferase reporter assays suggested that its methylation increases transcription. These results reveal a novel mechanism of gene expression whereby intragenic methylation can increase levels of transcription.

Alleles↗
PubMed2000

Igf2 imprinting in development and disease.

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.

Animals↗
PubMed1998

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes.

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

Alleles↗
PubMed1997

Conjunctival impression cytology in the preterm infant and it's relation to outcome.

UNLABELLED: The preterm infant is deficient in vitamin A (retinol) and this has been implicated in the pathogenesis of chronic lung disease of prematurity. Conjunctival impression cytology (CIC) has been used in adults to assess retinol status. We aimed to assess the feasibility of performing CIC in the preterm infant and to determine the significance of abnormal CIC findings. CIC samples were collected during routine retinopathy screening, and classified as inadequate, normal, borderline normal or abnormal. Ninety preterm infants were studied. Seventy-four (82%) CIC specimens produced a positive yield, whereas 16 (18%) were inadequate. Of the 74 adequate samples, 61 (82%) were normal or borderline normal and 13 (18%) abnormal. Seventy-three CIC specimens were assessed by a second histopathologist with complete agreement on 64 (88%) samples and disagreement on 9 (12%) samples. Ten sets of conjunctival impressions, taken from both eyes, gave identical results in all adequate samples. Birth weight was significantly lower in this abnormal group. Four infants (32%) in the abnormal group required treatment for retinopathy compared to two (3%) in the normal/borderline normal group, (P < 0.01). CONCLUSION: Conjunctival impression cytology is simple and reproducible technique which maybe easily applied to the preterm infant. Abnormal CIC is associated with retinopathy of prematurity requiring treatment.

Conjunctiva↗
PubMed1996

Randomised trial of effect of delayed intravenous lipid administration on chronic lung disease in preterm neonates.

A recent sevenfold increase in the annual incidence of chronic neonatal lung disease (CNLD) on an intensive care unit was attributed to the early administration of intravenous lipid (IVL) in ventilated preterm neonates. When logistic regression was used to eliminate other confounding variables, early delivery of IVL was independently associated with an eight-fold increase in the likelihood of CNLD. Consequently, we designed a prospective study to detect a halving of the incidence of CNLD by delaying IVL administration from 5 days (as is routine practice) to 14 days. Sixty-four parenterally fed preterm neonates weighing < 1,500 g at birth were randomised to receive IVL either on day 5 or day 14. Analysis was by intention to treat, since several infants in the latter group required no parenteral nutrition by day 14. Our results showed that the relative risk (95% confidence interval) of CNLD at 28 postnatal days was 1.15 (0.81-1.62); at 36 weeks postconception, it was 1.08 (0.59-1.99). A study population of > 2,000 would be required to determine whether these relative risks were significantly different from 1.

Chronic Disease↗
PubMed1996

Analysis of parent-specific gene expression in early mouse embryos and embryonic stem cells using high-resolution two-dimensional electrophoresis of proteins.

Genomic imprinting is an important genetic mechanism in mammals whereby certain genes are epigenetically modified and their expression altered according to their parental origin. The most important consequence of this is the requirement for both a maternal and a paternal genome for normal development to proceed to term. Although there are many instances of specific phenotypes (in the mouse) and diseases (in humans) resulting from imbalances in the parental chromosomes, it is only in the past few years that some of the imprinted genes responsible have been identified. It is however unclear what proportion of the genome is imprinted, particularly in the early embryo. To address the question to what extent parent-specific gene expression occurs in the early embryo and with a possible view to identifying new imprinted genes, the protein profiles of parthenogenetic and normal blastocysts were compared using the technique of high-resolution two-dimensional electrophoresis. The protein profiles of parthenogenetic, androgenetic and normal embryonic stem cells were also compared. Hence parent-specific gene expression was examined in embryonic and extraembryonic lineages of the early embryo. Approximately 1000 polypeptides were examined in each of the analyses, however no parent-specific differences were observed for any of these polypeptides. From this result, it is concluded that expression of genes encoding these polypeptides is identical from the parental chromosomes. These findings have important implications for estimates of the number of imprinted genes in the genome and for the interpretation of phenotypes of parthenogenetic and androgenetic embryos.

Animals↗
PubMed1979

Results of treatment of Ph'+ chronic myelogenous leukemia with an intensive treatment regimen (L-5 protocol).

Thirty-seven patients with Philadelphia-chromosone-positive (Ph'+) chronic myelogenous leukemia who were untreated or minimally pretreated were entered on the L-5 protocol. This protocol consisted of sequential treatment with splenic irradiation, splenectomy, arabinosylcytosine and 6-thioguanine, and L-asparaginase. Maintenance therapy was hydroxyurea or a multiple-drug regimen. The median survival of the 37 patients is 50 mo. Twelve patients showed a temporary reduction in the percentage of Ph'+ marrow metaphases to less than one-third of the initial values and in 7 of these patients none were found. The duration of the Ph'+ chromosome reduction ranged from 1 to 43 mo. The median survival of the responders has not yet been reached. It is concluded that whereas overall survival is not appreciably extended, patients who have a reduction in Ph'+ cells in the marrow may survive longer than the average; also, the reduction occurs most frequently in patients who have relatively small spleens at diagnosis. The reduction is difficult to maintain, and it may be reinduced in some patients with intensive chemotherapy.

Adolescent↗
PubMed1978

Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm.

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K(m) for acetyl-CoA of 10 mum and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K(i) 110 mum) and glycolate (K(i) 150 mum) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating (35)S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [(35)S]methionine-labeled endosperm tissue.

Journal Article↗
PubMed1978

Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum.

At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [(35)S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2-day-old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, (35)S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction.Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [(35)S]methionine or (14)C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes.Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes.

Journal Article↗
PubMed1976

Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm.

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.

Ricinus communis↗
PubMed1976

The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.

Ricinus communis↗
PubMed1975

Solitary bony metastasis as the first sign of malignant gastric tumor or of its recurrence.

Symptomatic solitary bony metastasis as the First sign of asymptomatic gastric carcinoma is very infrequent. Only 8 cases reported by 7 authors have been found in the literature. Furthermore, solitary bony metastasis as the sole sign of recurrence after hopefully curative resections are so rare that none has been previously reported in the literature. Three additional instances of solitary and histologically proven osseous metastasis of malignant gastric neoplasms have been observed and treated at Memorial Hospital during the years 1949 through 1969, and are herewith reported.

Adenocarcinoma↗
PubMed1974

Elective total gastrectomy for cancer of the stomach: end results.

There has been no apparent improvement in overall salvage of patients with cancer of the stomach treated by elective extended total gastrectomy from 1950-1964 as compared with those treated by partial gastrectomy during the preceding 20-year period, 1931-1950 at Memorial Hospital in New York City. Criteria of resectability have been extended in recent years, and, therefore, the two series of patients cannot be considered strictly comparable. In the 94 patients subjected to elective total gastrectomy for cancer of the stomach, more than half (55 patients) had cancer in the proximal 1/3 of the stomach. The results obtained in this group by total gastrectomy are inferior to those obtained in the earlier series by partial gastrectomy. Patients with carcinoma of the mid 1/3 of the stomach showed essentially the same 5-year survival by elective total gastrectomy (34.8%) as by partial gastrectomy (33.5%), while those with carcinoma of the distal 1/3 of the stomach showed a greater 5-year survival by elective total gastrectomy (43.7%) than by partial gastrectomy (29.8%). However, of significance is the fact that the incidence of nodal metastasis was 3 times greater in the patients undergoing elective total gastrectomy than in those undergoing partial gastrectomy. Despite this unfavorable finding, 5-year survival in the patients undergoing elective total gastrectomy for carcinoma of the mid 1/3 or distal 1/3 of the stomach was equal to, or better than, that found in those undergoing partial gastrectomy for lesions similarly located. On the basis of this finding alone, we believe that elective total gastrectomy is a worthwhile endeavor and should be performed for operable carcinomas arising in the mid 1/3 or distal 1/3 of the stomach.

Adult↗