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Biomedical subjects

R Tilly

Publications and source records attributed to R Tilly.

At least 19 recordsLinked to original sources
PubMed1988

Structural and functional characterization of genes encoding Dictyostelium prestalk and prespore cell-specific proteins.

The nucleotide sequence of D19, a Dictyostelium gene that encodes a prespore-specific mRNA sequence shows it to encode PsA, the cell surface protein detected by the MUD 1 monoclonal antibody. The predicted sequence of the protein reveals a largely hydrophobic C terminus, with chemical similarity to proteins known to be attached to the plasma membrane via a phosphatidylinositol link. The C-terminal region has direct sequence homology to the contact sites A protein and to the phosphatidylinositol-linked form of a chicken N-CAM, suggesting that it might play a role in cell adhesion. Expression of the D19 gene is known to be induced by cAMP and repressed by adenosine. The accumulation of the D19 mRNA is also repressed by DIF, the putative stalk-specific morphogen, and this effect is mediated at the transcriptional level. The pDd56 and pDd63 genes are induced by DIF, and they are specific markers of prestalk and stalk cells. They encode, respectively, ST310 and ST430, two proteins that were first identified by two-dimensional gel electrophoresis. Both proteins are predominantly composed of a highly conserved, 24-amino acid repeat. The two proteins are localized in the slime sheath of the migratory slug and in the stalk tube and stalk cell wall of the mature culminant, where they presumably function as structural components of the extracellular matrix. We have constructed marked derivatives of the pDd56, pDd63, and D19 genes, and these are correctly regulated after transformation into Dictyostelium cells. Thus we have determined the structure, and elucidated possible functions, for one prespore and two prestalk genes. These sequences should be of value, both as markers of the earliest events in cellular differentiation and in identifying the regulatory sequences controlling cell type-specific gene expression.

Amino Acid Sequence↗
PubMed1988

Identification and localization of proteins encoded by two DIF-inducible genes of Dictyostelium.

We show that pDd56 and pDd63, two related DIF-inducible genes of Dictyostelium, respectively encode the ST310 and ST430 polypeptides identified by Morrissey, Devine, and Loomis (1984, Dev. Biol. 103, 414-424). We localize the two proteins by immunoelectron microscopy to the extracellular matrix surrounding the stalk cells and the stalk tube. Coupled with their predicted amino acid sequence and biochemical properties, this suggests that they are structural proteins of the stalk.

Dictyostelium↗
PubMed1988

Cellular and molecular studies on infant null acute lymphoblastic leukemia.

We have studied the cellular and molecular basis of eight cases of infant null acute lymphoblastic leukemia (ALL). All eight patients were under 9 months of age and presented with leukocyte counts in excess of 60 X 10(9)/L, organomegaly, and in two cases CNS infiltration. Although seven cases were morphologically classified as ALL, one patient had both lymphoid and myeloid features. Phenotypic analysis of leukemic blasts from all patients showed a typical null ALL pattern, ie, CD10 (common ALL antigen)-negative, strongly HLA-DR-positive, and CD19 (B4)-positive. The presence of terminal deoxynucleotidyl transferase (TdT) at presentation was positive in six patients' cells and negative in two. Two patients also expressed the myeloid-associated markers CD33 (MY9) and CD15 (TG1), and coexpression of CD19 and CD33 was confirmed in these two by using dual marker flow cytometry (fluorescence-activated cell sorting). Electron microscopic examination of the same two patients' cells showed the presence of monocytoid blasts that labeled with the pan-B cell antibody B4 (CD19). Short-term culture of one of these patients cells in the presence of phorbol ester resulted in the majority of the cells exhibiting myeloid markers, strong nonspecific esterase positivity, and phagocytic properties. Cytogenetic analysis showed the common feature in 7 of 8 cases to be a break in band 11q23. Molecular analysis of DNA from the blast cells of all eight patients showed rearrangement of the immunoglobulin heavy-chain genes in all cases without, however, any evidence of kappa light-chain rearrangement. T cell receptor genes were present in the germline configuration in all cases. Rearrangements of the c-ets 1 oncogene, which maps to band 11q23, were not detected, thus providing no evidence for involvement of this oncogene in the common disease process. Our data indicate that although infant null ALL may present as a heterogeneous disease the similarity of many features between cases suggests a common derivation from a precursor cell sharing phenotypic and genotypic features of both B and myeloid progenitor cells.

Antigens, Neoplasm↗
PubMed1987

Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells.

Many existing MAbs raised against the human milk fat globule or against carcinoma cells recognise epitopes on high-molecular-weight glycoproteins. In a comparative ELISA assay a number of these antibodies have been shown to react with an extract of the human milk fat globule. In comparative immunoblots of cultured normal milk cells and breast cell lines, all were found to bind to large molecules which show some variation in molecular weight depending on the cell source. The HMFG-2 antibody, which is widely used in cancer diagnosis, also recognises epitopes on lower-molecular-weight components. In T47D cells these may be as small as 80,000 Mr, and with electron microscopy this cell line can be shown to accumulate HMFG-2-reactive components in the Golgi apparatus. Using an HMFG-2 affinity column we have immunopurified HMFG-2-reactive material from the 2 breast cancer cell lines MCF-7 and T47D and shown that all of the above antibodies react in a solid-phase ELISA with the purified material. In addition to high-molecular-weight components, the immunopurified material was found to contain lower-molecular-weight components including a glycoprotein of 68,000 Mr that was not, however, recognised by the HMFG-2 antibody on a Western blot. We have used this immunopurified material to generate new MAbs. All of these recognise the high-molecular-weight bands seen with the other antibodies, but 2 of them also recognise a band at 68,000 Mr in blots of MCF-7 and T47D. The second-generation antibodies show a spectrum of reactivity on tissues similar to HMFG-2 and one reacts at least as strongly as HMFG-2 with methanol-acetone-fixed sections of breast cancers.

Antibodies, Monoclonal↗
PubMed1987

Tissue-specific expression of the human type II collagen gene in mice.

Type II collagen is crucial to the development of form in vertebrates as it is the major protein of cartilage. To study the factors regulating its expression we introduced a cosmid containing the human type II collagen gene, including 4.5 kilobases of 5' and 2.2 kilobases of 3' flanking DNA, into embryonic stem cells in vitro. The transformed cells contribute to all tissues in chimeric mice allowing the expression of the exogenous gene to be studied in vivo. Human type II collagen mRNA is restricted to tissues showing transcription from the endogenous gene and human type II collagen is found in extracellular matrix surrounding chondrocytes in cartilage. The results indicate that the cis-acting requirements for correct temporal and spatial regulation of the gene are contained within the introduced DNA.

Animals↗
PubMed1987

Colonic pericrypt sheath cells: characterisation of cell type with new monoclonal antibody.

A new monoclonal antibody, PR 2D3, was raised against a crude homogenate of normal colorectal mucosa and found to react with the cells in the pericrypt sheath. It also reacted with smooth muscle throughout the body and, in specific sites, with those mesenchymal cells known as myofibroblasts. It did not react with cardiac or skeletal muscle, nor with fibroblasts. PR 2D3 is an IgG1 antibody and identifies a membrane component of about 140 K molecular weight. The pericrypt cells have been described as fibroblasts, but in view of the specificity of PR 2D3 for smooth muscle, and its selective staining of the colonic pericrypt cells, this cell type was re-examined for other smooth muscle properties. Ultrastructurally, the cells had many characteristics in common with smooth muscle and were identical with the myofibroblasts of the umbilical cord. On immunocytochemical examination they were found to contain desmin, myosin, and filamin. The confirmation that the pericrypt cells are myofibroblasts suggest that they have both contractile and secretory roles.

Animals↗
PubMed1983

Prostatic steroid-binding protein. Isolation and characterization of C3 genes.

Prostatic steroid-binding protein, whose expression is stimulated by androgens, consists of two subunits, one containing the polypeptides C1 and C3 and the other containing the polypeptides C2 and C3. We have isolated and sequenced cDNA clones specific for C3 mRNA and used them to isolate and characterize genomic clones for two C3 genes. Both genes are 3.2 kilobases with identical exon/intron arrangements, which is similar to the organization of the C1 and C2 genes, suggesting that they may have arisen by duplications of an ancestral gene. Finally, homologous human genes have not been detected.

Amino Acid Sequence↗
PubMed1983

A paralytic disease in nude mice associated with polyoma virus infection.

Nude mice (nu/nu), heterotransplanted with human tumours and kept in isolators, were found to suffer from wasting and posterior paralysis. Electron microscopy of spinal cord tissue revealed virus particles in the oligodendrocytes consistent in size (35 to 40 nm), morphology and distribution with those of the polyoma--SV40 sub-group of papovaviruses. Serology and restriction enzyme analysis of the virus genome showed that the virus was the murine polyoma A2 strain. Inoculation of uninfected nude mice with 10(7) TCID50 of polyoma A2 strain virus produced a similar disease in these mice with wasting and, after 10 to 23 weeks, paralysis of the hind legs of all surviving mice. Extensive myelin disruption was seen throughout the brain stem and sacral region of the spinal cord and high titres of polyoma virus were found in the whole brain (10(8.8) TCID50/brain) and in the spinal cord (10(6.8) TCID50/spinal cord).

Animals↗
PubMed1981

Cell interactions and endoderm differentiation in cultured mouse embryos.

Morphological and biochemical evidence is presented that the visceral extraembryonic endoderm of the 6.5-day mouse embryo will differentiate into parietal endoderm when cultured in contact with extraembryonic ectoderm undergoing transition into trophoblast giant cells. Egg cylinders from 6.5-day embryos were dissected into embryonic and extraembryonic halves and cultured in suspension in vitro for up to 7 days. After 4 days, the endoderm cells of the extraembryonic fragments morphologically resemble parietal endoderm, are associated with a thick basement membrane and synthesize large amounts of the matrix proteins laminin and Type IV procollagen. A similar transition in phenotype is not seen in the endoderm of embryonic fragments, nor in visceral extraembryonic endoderm cells cultured in isolation. In another series of experiments, complete egg cylinders were dissected free of visceral endoderm overlying the extraembryonic ectoderm and then cultured in vitro. The visceral endoderm cells which recolonize the surface of the extraembryonic ectoderm develop a parietal endoderm phenotype and lay down a thick basement membrane. These results suggest that the differentiation of the extraembryonic endoderm of the early mouse embryo into visceral and parietal phenotypes can be influenced by local cell-cell or cell-substrate interactions, and is not determined solely by cell lineage.

Animals↗
PubMed1980

Cell commitment and gene expression in the axolotl embryo.

In the axolotl embryo the somitic mesoderm passes through a reversible and then an irreversible phase of commitment with respect to its later differentiation into muscle. We show that the commencement of alpha-actin synthesis and the first appearance of thin myofilaments occur at the the same developmental stage as the transition between these phases. In intact embryos beta-and gamma-actin are made at all stages of development and in all tissues. alpha-actin, however, first appears at the late head process stage and is confined to the somites and tailbud, the regions of the embryo which later form the myotomal muscles. The thin filaments first appear near the myocoel of the somites in association with a novel organelle of mottled appearance and are quite distinct in structure from the epidermal microfilament networks found in embryos of the same stage. Cell fusion occurs somewhat later, although before the appearance of muscle striations in the light microscope. Well formed sarcomeres are not found until some time after the onset of motility. Presumptive somitic mesoderm isolated at a stage before the transition and cultured in a buffered salt solution for six days autonomously begins to synthesize alpha-actin and develop sarcomeres with the normal structure.

Actins↗
PubMed1980

Demyelination and wasting associated with polyomavirus infection in nude (nu/nu) mice.

Nude (nu/nu) mice bearing human tumour heterografts were affected with posterior paralysis and wasting. There was demyelination and infection of the oligodendrocytes of the spinal cord with a papovavirus. Similar virus particles and inclusion bodies were found in the bronchial epithelium, which showed histopathological changes. Similar changes were shown by the epithelia of the renal pelvis, ureter and choroid plexus. The virus was found in a transplantable human tumour, and evidence of spread by contact was also obtained. Intracerebral injection of spinal cord suspension from infected mice resulted in virus infected cutaneous carcinomata, demyelination with virus particles in the oligodendrocytes and posterior paralysis with wasting in adult nude mice. The suspension injected intraperitoneally into newborn Syrian hamsters produced tumours similar to those produced by murine polyoma. No evidence of infection was found in mice from the colony of origin. The virus was identified as murine polyoma Wild Type A2.

Animals↗
PubMed1978

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. II. Inner cell masses from 3.5- to 4.0-day p.c. blastocysts.

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3.5 and 4.0 days p.c. By 24--48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm-like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

Animals↗
PubMed1978

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. I. Inner cell masses from 3.5-day p.c. blastocysts incubated for 24 h before immunosurgery.

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3.5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7.5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4.5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

Animals↗