PubMed Health⌕ Search

Biomedical subjects

A A Mills

Publications and source records attributed to A A Mills.

17 recordsLinked to original sources
PubMed2007

p53: at the crossroad between cancer and ageing.

The p53 tumour suppressor plays an undisputed role in cancer. p53's tumour suppressive activity stems from its ability to respond to a variety of stresses to trigger cell cycle arrest, apoptosis or senescence, thereby protecting against malignant transformation. An increasing body of evidence suggests that p53 also drives organismal ageing. Although genetic models with altered p53 function display age-related phenotypes and thus provide in vivo evidence that p53 contributes to the ageing process, p53's role in organismal ageing remains controversial. Anti-cancer therapies that target p53 and reactivate or enhance its activity are considered good alternatives for treating various neoplasms. Therefore, it is important to determine whether these clinical approaches compromise tissue homeostasis and contribute to ageing. This review presents a number of models with altered p53 function and discusses how these models implicate p53 as part of a molecular network that integrates tumour suppression and ageing.

Aging↗
PubMed2001

Introducing defined chromosomal rearrangements into the mouse genome.

Chromosomal rearrangements have been instrumental in genetic studies in Drosophila. Visibly marked deficiencies (deletions) are used in mapping studies and region-specific mutagenesis screens by providing segmental haploidy required to uncover recessive mutations. Marked recessive lethal inversions are used as balancer chromosomes to maintain recessive lethal mutations and to maintain the integrity of mutagenized chromosomes. In mice, studies on series of radiation-induced deletions that surround several visible mutations have yielded invaluable functional genomic information in the regions analyzed. However, most regions of the mouse genome are not accessible to such analyses due to a lack of marked chromosomal rearrangements. Here we describe a method to generate defined chromosomal rearrangements using the Cre--loxP recombination system based on a published strategy [R. Ramirez-Solis, P. Liu, and A. Bradley, (1995) Nature 378, 720--724]. Various types of rearrangements, such as deletions, duplications, inversions, and translocations, can be engineered using this strategy. Furthermore, the rearrangements can be visibly marked with coat color genes, providing essential reagents for large-scale recessive genetic screens in the mouse. The ability to generate marked chromosomal rearrangements will help to elevate the level of manipulative mouse genetics to that of Drosophila genetics.

Animals↗
PubMed2001

From mouse to man: generating megabase chromosome rearrangements.

Experimental approaches for deciphering the function of human genes rely heavily on our ability to generate mutations in model organisms such as the mouse. However, because recessive mutations are masked by the wild-type allele in the diploid context, conventional mutagenesis and screening is often laborious and costly. Chromosome engineering combines the power of gene targeting in embryonic stem (ES) cells with Cre--loxP technology to create mice that are functionally haploid in discrete portions of the genome. Chromosome deletions, duplications and inversions can be tagged with visible markers, facilitating strain maintenance. These approaches allow for more refined mutagenesis screens that will greatly accelerate functional mouse genomics and generate mammalian models for developmental processes and cancer.

Animals↗
PubMed2000

Outcome of pregnancies complicated by ruptured membranes after genetic amniocentesis.

OBJECTIVE: We sought to compare perinatal outcomes of pregnancies complicated by preterm premature rupture of membranes after genetic amniocentesis with pregnancies complicated by spontaneous preterm premature rupture of membranes at a similar gestational age. STUDY DESIGN: A retrospective study was performed in which a computerized database was reviewed to identify all patients presenting to our institution with preterm premature rupture of membranes within 48 hours of a genetic amniocentesis from July 1988 to August 1999. Control subjects were matched for gestational age at preterm premature rupture of membranes. Patients were all managed expectantly. Outcomes were compiled from review of medical records. Descriptive statistics, the Student t test, and the chi(2) test were used, with P <.05 considered significant. RESULTS: During the study period, genetic amniocentesis was performed 1101 times. Eleven (1%) women presented within 48 hours with preterm premature rupture of membranes. The mean gestational age at the time of rupture was not different between the cases in which preterm premature rupture of membranes occurred after genetic amniocentesis compared with the control subjects in whom preterm premature rupture of membranes occurred spontaneously (16.5 weeks vs 17.6 weeks, respectively). Women with preterm premature rupture of membranes after amniocentesis experienced significantly longer latency periods (124 vs 28 days; P =.0001) and delivered at more advanced gestational ages (34.2 vs 21.6 weeks; P =.0002) than those with spontaneous preterm premature rupture of membranes. The perinatal survival rate was 91% in pregnancies complicated by preterm premature rupture of membranes after genetic amniocentesis compared with a rate of 9% in control subjects (P =.005). CONCLUSIONS: Pregnancies complicated by preterm premature rupture of membranes after genetic amniocentesis result in significantly better perinatal outcomes compared with pregnancies complicated by spontaneous preterm premature rupture of membranes at a similar gestational age. Expectant management should be considered in such cases.

Adult↗
PubMed1999

A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice.

Gene targeting in mouse embryonic stem (ES) cells can be used to generate single gene mutations or defined multi-megabase chromosomal rearrangements when applied with the Cre- loxP recombination system. While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearrangements are great genetic tools for mapping, mutagenesis screens and functional genomics. The conventional approach to generate mice with targeted alterations of the genome requires extensive molecular cloning to build targeting vectors and DNA-based genotyping for stock maintenance. Here we describe the design and construction of a two-library system to facilitate high throughput gene targeting and chromo-somal engineering. The unique feature of these libraries is that once a clone is isolated, it is essentially ready to be used for insertional targeting in ES cells. The two libraries each bear a complementary set of genetic markers tailored so that the vector can be used for Cre- loxP -based chromosome engineering as well as single knockouts. By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice with single gene knockouts or more extensive chromosomal rearrangements.

Agouti Signaling Protein↗
PubMed1999

p63 is a p53 homologue required for limb and epidermal morphogenesis.

The p53 tumour suppressor is a transcription factor that regulates the progression of the cell through its cycle and cell death (apoptosis) in response to environmental stimuli such as DNA damage and hypoxia. Even though p53 modulates these critical cellular processes, mice that lack p53 are developmentally normal, suggesting that p53-related proteins might compensate for the functions of p53 during embryogenesis. Two p53 homologues, p63 and p73, are known and here we describe the function of p63 in vivo. Mice lacking p63 are born alive but have striking developmental defects. Their limbs are absent or truncated, defects that are caused by a failure of the apical ectodermal ridge to differentiate. The skin of p63-deficient mice does not progress past an early developmental stage: it lacks stratification and does not express differentiation markers. Structures dependent upon epidermal-mesenchymal interactions during embryonic development, such as hair follicles, teeth and mammary glands, are absent in p63-deficient mice. Thus, in contrast to p53, p63 is essential for several aspects of ectodermal differentiation during embryogenesis.

Animals↗
PubMed1999

Analysis of the pattern of QM expression during mouse development.

QM, a novel gene that was originally identified as a putative tumor suppressor gene, has since been cloned from species encompassing members of the plant, animal, and fungal kingdoms. Sequence comparison indicates that QM has been highly conserved throughout eukaryotic evolution. QM is a member of a multigene family in both mouse and man, is expressed in a broad range of tissues, and is downregulated during adipocyte differentiation. Jif-1, a chicken homolog of QM, has been reported to interact with the protooncogene c-Jun, and to inhibit transactivation of AP-1 regulated promoters in vitro. Furthermore, disruption of the yeast QM homolog is lethal. Although these studies suggest that the QM gene product plays an important role within the normal cell, the precise role of QM has remained elusive. In this study, a thorough analysis of the pattern of QM expression during mouse development was undertaken, using the techniques of whole mount in situ hybridization and whole mount immunohistochemistry, in combination with conventional immunohistochemical analysis of tissue sections. QM is expressed in numerous embryonic tissues, and is differentially expressed throughout the embryo. The cytoplasmic localization of QM is consistent with its reported association with ribosomes, and inconsistent with its previously hypothesized function as a direct modulator of the nuclear protooncogene c-Jun. QM is expressed in the developing epidermis, and is particularly strong within developing limbs. Analysis of embryos of various stages of gestation indicate that QM is downregulated in the surface ectoderm of the embryo as development proceeds. QM protein is not detectable within either nucleated or enucleated red blood cell precursors. QM is strongly expressed within chondrocytes within the transition zone of developing limb cartilage, as well as within differentiated keratinocytes of the suprabasal regions of the epidermis. Furthermore, within both cartilage and skin, there is an inverse relationship between QM expression and proliferative capacity. This pattern of QM expression suggests that this novel gene product may be involved in processes such as posttranslational protein processing which are essential for differentiation of specific tissues during embryogenesis.

Animals↗
PubMed1998

Assembly of the QM protein onto the 60S ribosomal subunit occurs in the cytoplasm.

QM is a human cDNA originally isolated as a transcript elevated in a nontumorigenic Wilms' tumor microcell hybrid, relative to the tumorigenic parental cell line. The QM gene encodes a 24 kDa basic protein that peripherally associates with the ribosomes. Recently, the gene for this protein has also been shown in Saccharomyces cerevisiae to encode an essential 60S ribosomal subunit protein that is required for the joining of the 40S and 60S subunits. Since the association of QM with ribosomes can be disrupted with 1M NaCl, which has no effect on the association of core ribosomal proteins, indirect immunofluorescent cell staining was performed to colocalize the QM protein with the human large P-antigen, a core ribosomal protein of the 60S subunit, and to determine whether the assembly of the QM protein onto the 60S ribosomal subunit occurs in the nucleolus or in the cytoplasm. Our results reveal that QM co-localizes with the large P-antigen only to the cytoplasm where the rough endoplasmic reticulum is found and not to the nucleolus where ribosome assembly occurs. This finding suggests that the QM protein is most likely involved in a late step of the 60S subunit assembly and is added to the 60S ribosomal subunit in the cytoplasm and not in the nucleolus.

Antigens↗
PubMed1994

Extreme evolutionary conservation of QM, a novel c-Jun associated transcription factor.

QM is a 214 amino acid polypeptide, encoded by a gene (DXS648) in Xq28, that contains a high percentage of charged amino acids and has been found to bind c-Jun and DNA. Searches of the GenBank database revealed no matches between QM and any other known transcription factors. However, we and others have isolated QM homologs from a diverse array of eukaryotes. Alignment of these sequences indicated a high degree of conservation throughout the first 175 residues of the protein and revealed several interesting features. Most notable is the considerable conservation of charged amino acids within specific regions of the protein. Secondary structure analysis suggests that two of these regions form amphipathic alpha-helices, one basic and one acidic. A third conserved charged domain, comprising the N-terminal 30 amino acids, is both basic and proline rich. The rate of sequence divergence of the various homologs was found to be slow (of the order of 1% change every 22 million years), consistent with a critical role for QM in eukaryotic cells. A role for QM as a novel class of transcription regulatory protein is suggested.

Amino Acid Sequence↗
PubMed1994

Trypanosoma cruzi glycoprotein of M(r) 56,000 characterization and assessment of its potential to protect against fatal parasite infections.

A approximately 56,000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin-fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glycoprotein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10,000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi-subunit vaccine against T. cruzi.

Animals↗
PubMed1993

Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi.

The major proteolytic activity of Trypanosoma cruzi is a cathepsin L-like cysteine protease expressed in all stages of the parasite. As an initial step in identifying possible functions of this enzyme in the life cycle of T. cruzi, and examining its potential as a target for rational drug design, two fluoromethyl ketone-derivatized cysteine protease inhibitors were studied for their effects on T. cruzi infection of mammalian cells. Both inhibitors are irreversible substrate analogues with high specificity for cysteine proteases and minimal toxicity to mammalian cells. While micromolar concentrations of inhibitors had some effect on replication of all parasite stages, the most dramatic arrest of parasite replication occurred at the transformation of trypomastigote to amastigote, and also from amastigote to trypomastigote. It is therefore proposed that the enzyme functions in intracellular protein degradation in some stages of T. cruzi, but also in remodeling of the parasite during transformation between stages. Concentrations of inhibitors necessary to interrupt the parasite life cycle had no observable toxicity to macrophages, fibroblasts or epithelial cells in culture. Differential susceptibility of T. cruzi versus host cysteine proteases to fluoromethyl ketone protease inhibitors suggests that inhibition of the T. cruzi cysteine protease is a potential lead for new chemotherapy of Chagas' disease.

Amino Acid Chloromethyl Ketones↗
PubMed1992

The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi.

The complete sequence of the gene encoding the major cysteine protease from Trypanosoma cruzi is reported. The amino acid sequence predicted from the gene sequence aligns well with members of the papain family of cysteine proteases, suggesting the name cruzain. The sequence is most closely related to the cysteine protease of Trypanosoma brucei (59.3%) and the murine cathepsin L (42.2%). At least six copies of the gene are present in the genome and are organized in a tandem array of copies which are identical in all restriction endonuclease sites tested. The gene appears to be expressed in all developmental stages of T. cruzi with mRNA levels approximately 2-fold higher in the intracellular amastigote form. A copy of the T. cruzi gene was expressed in bacteria as an inactive, insoluble fusion polypeptide to approximately 5% of the total cell protein. The fusion protein was readily purified, solubilized in urea, and successfully refolded to produce a polyprotein which processed autocatalytically to yield approximately 1 mg of active protease per 3 g of wet cell paste. The processed form of the recombinant protease has an NH2-terminal sequence identical to that of the mature form of the protease purified from T. cruzi (Murta, A. C. M., Persechini, P. M., Souto-Padrón, T., de Souza, W., Guimaraes, J. A., and Scharfstein, J. (1990) Mol. Biochem. Parasitol. 43, 27-38; Cazzulo, J. J., Couso, R., Raimondi, A., Wernstedt, C., and Hellman, U. (1989) Mol. Biochem. Parasitol. 33, 33-42). This suggests that the recombinant protease possesses the requisite specificity and activity to correctly process the proform of the protease in vitro. Kinetic assays with peptide substrates demonstrate that the substrate specificity and kinetic parameters for the recombinant protease are consistent with those of the endogenous protease. The proteolytic activity of the recombinant protease is enhanced by dithiothreitol, inhibited by leupeptin, N alpha-p-tosyl-L-lysine chloromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) but is unaffected by phenylmethylsulfonyl fluoride, pepstatin, and 1,10-phenanthroline. More specifically, the recombinant enzyme was inhibited by benzyloxycarbonyl-Phe-Arg-fluoromethyl ketone, which inhibits replication and differentiation of T. cruzi within mammalian cells in culture.

Amino Acid Sequence↗
PubMed1992

Trypanosoma cruzi glycoprotein 72: immunological analysis and cellular localization.

Two monoclonal antibodies were used to biochemically characterize glycoprotein 72 (GP72) from Trypanosoma cruzi and to localize the protein in live and fixed parasites by indirect immunofluorescence and in thin section of parasites by immunogold electron microscopy. GP72 was shown in immunoblots to be specific for the epimastigote stage; the protein could not be detected in trypomastigotes. Each antibody reacted with a different epitope on the glycoprotein and deglycosylation of GP72 ablated reactivity with one of the antibodies. Indirect immunofluorescence and electron microscopic evaluation of parasite associated gold particles showed the presence of GP72 in the cell surface membrane including the flagellar pocket and the cytostome. In addition, cytoplasmic membrane vesicles of the endosomal-lysosomal system stained intensely.

Animals↗
PubMed1991

Canada balsam.

Explore the source record for details and available documents.

Canada↗