Epithelial carcinogenesis in the mouse: correlating the genetics and the biology.

Tumour formation relies on a complex combination of genetic and environmental factors. In particular, the contributions from inherited predisposition genes as well as carcinogens, for example from cigarettes or in the diet, are amongst the major contributors to tumorigenesis. Since the study of such processes in particularly difficult in human cancers, the availability of a well-defined model system is of obvious benefit. The mouse skin model of multistage carcinogenesis offers an excellent tool for the study of the target cells, the target genes and the biological events associated with neoplasia. In this system, tumorigenesis occurs in a series of defined stages, each of which is characterized by specific and reproducible alterations in genes such as H-ras, cyclin D1, p53 and p16INK4A. Additional changes occur in the production of, or response to, factors such as transforming growth factor beta (TGF beta). These genetic and biological alterations are mirrored in human tumours of epithelial origin. Hence, research into the general principles of tumour initiation, promotion and progression in the context of the mouse skin model is likely to prove valuable in the continual search for new methods for the diagnosis, prevention, and therapeutic treatment of human cancers.     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

Gibberellin-sensitive Suspension Cultures

Suspension cultures were incubated in the presence and absence of gibberellic acid (GA₃) in an attempt to define a new experimental system for study of the molecular action of gibberellins upon growth. Unlike many suspension cultures, an auxin-independent green clone from spinach (Spinacia oleracea L.) and an auxin-dependent line of “Paul's Scarlet” rose (Rosa sp.) were promoted in expansion growth by GA₃ at 10⁻¹¹ to 10⁻⁶ molar. In Rosa the cells also elongated upon GA₃ treatment whereas in Spinacia they remained isodiametric.     

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.     

Depletion of CD4+ (L3T4+) lymphocytes in vivo impairs murine host defense to Cryptococcus neoformans

T cell-mediated immunity has been shown to play an important role in the host defense to Cryptococcus neoformans. Infections due to C. neoformans are increased in patients with AIDS who are deficient in the CD4+ subset of T lymphocytes. Thus, the effect of CD4+ (L3T4+) lymphocyte depletion on murine host defenses to C. neoformans was studied. The mAb GK 1.5 was administered to mice, and CD4+ T lymphocyte depletion was confirmed by the analysis of T cell subsets in blood, spleen, lymph node, and lung. Evidence of a functional defect was confirmed by demonstrating that the splenocytes of treated mice were unable to proliferate in response to class II incompatible spleen cells. Furthermore, delayed type hypersensitivity to C. neoformans was abrogated by CD4+ lymphocyte depletion. Mice depleted of CD4+ lymphocytes were inoculated with a virulent strain of C. neoformans by the i.v. or the intratracheal route. After i.v. inoculation of C. neoformans, the survival of mice depleted of CD4+ lymphocytes was reduced (27.8 +/- 1.8 vs 36.0 +/- 3.1 days, p less than 0.04). After intratracheal inoculation, C. neoformans disseminated from the lung to extrapulmonary organs. Dissemination occurred earlier in mice depleted of CD4+ lymphocytes compared to mice that received control antibody, and the burden of C. neoformans in extrapulmonary organs was greater in mice depleted of CD4+ lymphocytes than control mice. Surprisingly, there was no increase in the burden of C. neoformans in the lungs of CD4+ lymphocyte-depleted mice. Survival of mice inoculated with C. neoformans and depleted of CD4+ lymphocytes was reduced compared to control mice and was related to the increased rate of accumulation of organisms in the brains of treated mice. The mean survival of GK 1.5-treated mice was 34.1 +/- 0.9 days compared to control mice with a mean survival of 40.6 +/- 9 days (p less than 0.001). These data suggest that CD4+ lymphocytes play a prominent role in the host defense of infections due to C. neoformans, that CD4+ lymphocytes are required in extrapulmonary organs for optimal clearance of C. neoformans and that CD4+ lymphocytes are critical for survival of mice infected with C. neoformans.     

Domains of receptor mobility and endocytosis in the membranes of neonatal human erythrocytes in the membranes of neonatal human erythrocytes and reticulocytes are deficient in spectrin

It has previously shown (Schekman, R., and S.J. Singer, Proc. Natl. Acad. Sci. U.S.A. 73:4075-4079) that receptors in the membranes of neonatal human erythrocytes show a restricted degree of lateral mobility, whereas in adult human erythrocytes the receptors are essentially immobile. This restricted mobility is exhibited, for example, when concanavalin A (Con A) induces a limited clustering of its receptors in the neonatal erythrocyte membrane, resulting in the formation of invaginations and endocytic vesicles. This does not happen with adult cells. By the use of indirect immunoferritin labeling of ultrathin frozen sections of Con A-treated neonatal blood cells, we now show that the invaginations and endocytotic vesicles do not stain for spectrin, whereas the adjacent unperturbed membrane is heavily stained. The reticulocytes in the neonatal cell population undergo substantially more Con A-induced invagination and endocytosis than do the erythrocytes. These results lend strong support to the hypothesis that specialized discrete domains exist, or are induced, in the membranes of these neonatal cells, in which receptors are laterally mobile, whereas in the remaining (and predominant) part of the membrane the receptors are immobile. Such mobile domains are characterized by an absence of spectrin. During the maturation of the neonatal reticulocyte to erythrocyte, it is proposed that these domains are in large part, but not completely, eliminated.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

Anatomical and gravitational influences on cardiac displacement in snakes (Lepidosauria,Serpentes)

Summary Radiographs of live, unanesthetized snakes were used to document the position of the heart in the body cavity during horizontal, head-up, and head-down postures. The extent of cardiac displacement observed during these postural changes differed substantially among the snakes examined, ranging from virtually none in a thin-bodied arboreal snake to as much as three vertebral lengths (=half the length of the heart) in a heavy-bodied terrestrial Crotalus. The basis of this differential cardiac displacement is attributed to the anatomical packaging of the pericardial sac. In some snakes the pericardial sac is loosely suspended in the body cavity by the great vessels and connective tissue sheets. In contrast, in other snakes the pericardial sac is buttressed against the body wall, the lung, or the liver. We hypothesize that cardiac displacement during postural change may alter the pattern of blood flow in the aortae of snakes.