Profile. Telling Ken to stop the rot. Interview by Adrian O'Dowd.
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Humans↗
Biomedical subjects
T Sanford
Publications and source records attributed to T Sanford.
Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.
The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.
Actinin↗
Identification of nine novel mutations in cartilage oligomeric matrix protein in patients with pseudoachondroplasia and multiple epiphyseal dysplasia.
Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are allelic disorders caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). PSACH is a dominant condition characterized by disproportionate short stature, joint laxity, and early-onset osteoarthritis. EDM1 is a less severe skeletal dysplasia associated with average to mild short stature, joint pain, and early-onset osteoarthritis. COMP is an extracellular matrix protein present in cartilage, ligament, and tendon tissues. Here, we report on nine novel mutations in COMP causing PSACH and EDM1. Four of these mutations are in exons 13C and 14 where no previous mutations had been reported. One of those mutations was identified in two separate EDM1 families. In addition, we have identified the first case of PSACH resulting from an expansion of the five aspartates in exon 17B. We are also reporting a mutation in a third PSACH family with somatic/germline mosaicism. Therefore, this report increases the range of mutations that cause PSACH and EDM1 and provides additional regions to target for mutational analysis.
Achondroplasia↗
Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.
Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.
Animals↗
Characterization of human papillomavirus in airway papillomas by histologic and biochemical analysis.
The role of human papillomavirus (HPV) in airway papillomas has been well defined in recent literature. The chronicity and recurrence of papillomas has been postulated to be a result of residual viral genome in tissue treated with standard laser techniques. Thirteen patients with airway papillomas were selected for study with polymerase chain reaction (PCR) methods to detect viral DNA. Specimens taken prior to laser therapy and specimens taken at laser margins were consistently positive for HPV DNA by PCR. The HPV DNA is apparently present in tissues after macroscopic disease has been ablated by laser techniques. Histologic analysis of laser biopsies demonstrated fragments of squamous epithelium with cytologic features of HPV infection. Laser treatment is ineffective in eradicating HPV-infected tissues from airway papillomas, and this finding supports the notion that recurrence is a product of HPV incorporated into tissue not ablated by laser irradiation. Specific methods, results, and clinical correlation will be discussed.
Adult↗
Identification of twelve mutations in cartilage oligomeric matrix protein (COMP) in patients with pseudoachondroplasia.
Pseudoachondroplasia (PSACH) is an autosomal dominant dwarfing condition characterized by disproportionate short stature, joint laxity, and early-onset osteoarthrosis. PSACH is caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). We are reporting on mutations in COMP in 12 patients with PSACH, including ten novel mutations. Eleven of the mutations are in exons 17A, 17B, and 18A, which encode the calcium-binding domains, and one mutation is in exon 19, which encodes part of the carboxy-terminal globular domain. Two of the mutations identified are the common delGAC(1430-1444) in exon 17B, which accounts for 36% of identified PSACH mutations. This report increases the range of mutations in COMP that cause PSACH and provides additional evidence for the importance of the calcium-binding domains and the globular domain to the function of COMP.
Achondroplasia↗
Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing.
We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ubiquitous nuclear enzyme that converts multiple adenosines to inosines in double-helical RNA substrates without apparent sequence specificity. The A --> I conversion activity of the protein encoded by the cloned cDNA was confirmed by recombinant expression in insect cells. Use of the cloned DNA as a molecular probe documented sequence conservation across mammals and detected a single transcript of 7 kb in RNA of all human tissues analyzed. The deduced primary structure of human DRADA revealed a bipartite nuclear localization signal, three repeats of a double-stranded RNA binding motif, and the presence of sequences conserved in the catalytic center of other deaminases, including a cytidine deaminase involved in the RNA editing of apolipoprotein B. These structural properties are consistent with the enzymatic signature of DRADA, and strengthen the hypothesis that DRADA carries out the RNA editing of transcripts encoding glutamate-gated ion channels in brain.
Adenosine Deaminase↗
Purification and characterization of double-stranded RNA adenosine deaminase from bovine nuclear extracts.
The double-stranded RNA (dsRNA) adenosine deaminase (DRADA) deaminates adenosine residues to inosines and creates I-U mismatched base pairs in dsRNAs. Its involvement in RNA editing of glutamate-gated ion channel gene transcripts in mammalian brains has been proposed as one of the biological functions for this recently identified cellular enzyme. We purified a mixture of three forms, 93, 88, and 83 kDa, of bovine DRADA proteins, all likely to be active enzymes. We determined that DRADA has a native molecular mass of approximately 100 kDa, suggesting that the enzyme exists as a monomer. The purified enzyme was not inhibited by 2'-deoxycoformycin, a transition state analog inhibitor of adenosine deaminase and AMP deaminase, suggesting that the catalytic mechanism of DRADA might be different from that of other deaminases. DRADA binds specifically to dsRNA with a dissociation constant of 0.23 nM for a synthetic dsRNA, and the Michaelis constant is 0.85 nM. These values indicate that DRADA has a much higher affinity for its substrate than other deaminases such as adenosine deaminase and AMP deaminase. DRADA may need this extremely high affinity to catalyze efficiently the modification of relatively rare substrate RNAs in the cell nucleus.
Adenosine Deaminase↗
Relationship between macrophage colony-stimulating factor production by uterine epithelial cells and accumulation and distribution of macrophages in the uterus of pregnant mice.
Estrogen and progesterone induce production of macrophage colony-stimulating factor (CSF-1) by uterine epithelial cells, and CSF-1 is produced in the uterus during pregnancy in mice. CSF-1 is a lineage-specific stimulator of macrophage proliferation, chemotaxis, and function. High concentrations of macrophages accumulate in the uterus during pregnancy. Experiments were conducted to determine whether a relationship exists between intrauterine CSF-1 production and the number and distribution of uterine macrophages during pregnancy in mice. The study demonstrated that on day 1 of pregnancy CSF-1 bioactivity levels were high. The number of macrophages in the uterus was also high on days 1 and 2, and macrophages were concentrated at epithelial surfaces. The decrease in CSF-1 bioactivity seen between days 1 and 2 was followed by a decrease in the macrophage concentration. An increase in CSF-1 bioactivity on day 4 was followed by an increase in the concentration of intrauterine macrophages. During the immediate postimplantation period, macrophages were detected primarily in the myometrium and deep endometrium and CSF-1 bioactivity was undetectable. During the second half of pregnancy, when CSF-1 concentrations were very high, the macrophage concentration was also very high and large numbers of macrophages were detected in association with epithelia. The data confirmed the existence of a direct relationship between intrauterine CSF-1 and macrophage accumulation and suggested that macrophages are attracted to epithelial surfaces by CSF-1.
Animals↗
Macrophage colony stimulating factor controls macrophage recruitment to the cycling mouse uterus.
Progesterone and estrogen induce uterine epithelial cells to produce macrophage colony stimulating factor (CSF-1), and mouse uterine epithelial cells produce large amounts of CSF-1 during embryo development. The present study demonstrated that estrogen and progesterone each induce uterine CSF-1 gene transcription and translation detectable by Northern blotting and bioassay. Intrauterine CSF-1 production was greater in the presence of both estrogen and progesterone than following exposure to either hormone alone. CSF-1 mRNA was detectable in the uterus throughout the estrous cycle while CSF-1 bioactivity was detected only during proestrus. CSF-1 production was directly related to the presence of macrophages in the uterus. Ovariectomy, which was rapidly followed by a loss of uterine CSF-1 gene transcription, also was followed by a dramatic decrease in the number of uterine macrophages. Both uterine CSF-1 and uterine macrophages were reconstituted in a dose-dependent manner by systemic administration of estrogen or progesterone. High concentrations of circulating estrogen and progesterone increased the number of macrophages in the uterus and increased their accumulation near epithelial surfaces. Similar relationships were observed in the uterus of cycling mice. Macrophages accumulated in the uterus following intraluminal injection of recombinant human CSF-1 to ovariectomized mice, directly demonstrating the ability of CSF-1 to recruit macrophages from peripheral blood into the uterus. These studies demonstrated that estrogen and progesterone stimulation of CSF-1 production by mouse uterine epithelial cells controls recruitment and distribution of macrophages in the uterus during the estrous cycle.
Animals↗
The days ahead.
How are government controls affecting healthcare foodservices?Are growing elderly & outpatient populations changing foodservices' missions. What are the hottest trends? As members of the American Society for Hospital Food Service Administrators prepared for their annual meeting in Orlando, FM queried 11 Southeastern directors to learn their most pressing concerns & how they are positioning their departments for the future.
Aged↗
Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia coli.
The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase. It was cloned at the polylinker region of pUC18 vector with Escherichia coli JM109 as the host. A clone was serendipitously isolated that expressed in E. coli a protein of approximately 120 kDa in size at high levels. DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF. Polyclonal antibodies raised against this protein purified from E. coli were used for immunological analysis. The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots. In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase alpha from uninfected HeLa cells. The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus. The replicating viral DNA and the polymerase were colocalized in these globular sites. Furthermore, HeLa cells infected with Ad5ts149, a temperature-sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.
Adenoviruses, Human↗
Purification and immunological analysis of RNA polymerase II from Caenorhabditis elegans.
We describe a rapid procedure for obtaining highly purified RNA polymerase II from the nematode Caenorhabditis elegans. The structure of the enzyme was examined by denaturing gel electrophoresis and found to consist of three large polypeptides (molecular weights 200,000, 175,000, and 135,000) and eight smaller polypeptides (molecular weights 29,500, 20,000, 16,000, 15,000, 13,000, 11,500, 10,500, and 9,500). As observed for the analogous enzyme from other organisms, the 175,000 polypeptide (II175) appeared to be a degraded form of the 200,000 polypeptide (II200). The structure of nematode RNA polymerase II closely resembles that of the corresponding enzyme from other animals. Four of its larger subunits shared antigenicity with Drosophila RNA polymerase II. Antibody raised against purified RNA polymerase II reacted with several enzyme subunits in "Western" blots of purified polymerase and impure enzyme fractions. Immunofluorescence staining was used to visualize RNA polymerase II in the nuclei of a nematode squash preparation and the nucleoplasm of cultured mammalian cells.
Animals↗
RNA polymerase II from wild type and alpha-amanitin-resistant strains of Caenorhabditis elegans.
DNA-dependent RNA polymerases I, II, and III have been isolated from the soil nematode, Caenorhabditis elegans, and RNA polymerase II has been partially purified. The sensitivities of these enzymes to alpha-amanitin resemble those of the cognate enzymes from vertebrates. RNA polymerase II from C. elegans is 50% inhibited by 7 ng/ml of the amatoxin and RNA polymerase III by 80 micrograms/ml, whereas RNA polymerase I is insensitive to 500 micrograms/ml. We have obtained mutants of C. elegans which can grow and reproduce in concentrations of alpha-amanitin which arrest development of wild type animals. One of these mutants (DR432) has an altered RNA polymerase II which in partially purified extracts is 150 times less sensitive to the drug than the wild type enzyme. The mutation, ama-1(m130), in DR432 is dominant and maps near dpy-13 on linkage group IV. RNA polymerase II isolated from ama-1/+ heterozygotes contains equal proportions of two components, corresponding in alpha-amanitin sensitivity to the enzymes from DR432 and wild type. Thus, ama-1 appears to affect a subunit of RNA polymerase II.
Amanitins↗
Alan Gregg Memorial Lecture. Educating the public about health. The role of formal education.
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Attitude to Health↗
Mechanism of lipid peroxidation in cancer cells in response to gamma-linolenic acid (GLA) analyzed by GC-MS(I): Conjugated dienes with peroxyl (or hydroperoxyl) groups and cell-killing effects.
We have investigated the nature of lipid peroxidation occurring in association with cancer-killing produced by gamma-linolenic acid (GLA) and iron (Fe) in cultured human breast cancer cells (ZR-75-1: ZR). UV-spectrophotometry, high performance liquid chromatography (HPLC) and gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) have been used to analyze lipid peroxides and their derivatives. Formation of conjugated dienes (CD), the conversion of triphenylphosphine (TPP) to its oxide (TPPO), and the simultaneous production of hydroxy polyunsaturated fatty acids (PUFA-OHs) from these corresponding PUFAs hydroperoxides (PUFA-OOHs) were analyzed in the total lipid extract of ZR cells and of normal human skin fibroblasts (CCD-41Sk:Sk). Fe enhanced the formation of both the CD and TPPO and increased the percentage of dead cells, while vitamin E inhibited these effects. Neither of these events was observed at any significant level in Sk cells. Identity of PUFA-OHs was confirmed by determining the regional positions of the hydroxyl group by GC-MS analysis of hydrogenated methyl ester tert-butyldimethylsilyl ether alcohol derivatives (Me-H2-PUFA-O-TBDMS). The regional isomers identified were 15-, 12- and 8-OH 20 carbon and 13-OH 18 carbon fatty acid derivatives. These results suggest that the increased formation of conjugated dienes and/or hydroperoxyl (or peroxyl) groups in PUFA molecules is relevant to the cancer cell-killing effect of GLA+Fe.
Breast Neoplasms↗