MEASUREMENT OF THE DURATION OF DNA SYNTHESIS IN ERYTHROID CELLS OF THE BONE MARROW USING A DOUBLE LABELLING AUTORADIOGRAPHIC TECHNIQUE DESIGNED SPECIFICALLY FOR USE WITH HAEMOPOIETIC TISSUES

This paper describes a double labelling autoradiographic technique for use with haemopoietic tissues. In involves two photographic emulsions separated by a thin piece of mica on which the cells have been smeared. In this way the autoradiographic grains due to tritium and carbon-14 appear above and below the cells respectively. Applying the method to bone marrow normoblasts of young rats, the average duration of DNA synthesis (t_s) for the pro- and early normoblasts taken together is found to be 5.1 hr and the mean cell cycle time (t_c) to be 8.2 hr. For the intermediate normoblasts, the corresponding figures are 6.3 hr and 15.7 hr. Average values for all dividing normoblasts in the bone marrow are 5.8 hr and 12.8 hr respectively for t_s and t_c. The average duration of mitosis is 32 min.     

Immunotoxins: status and prospects.

Immunotoxins, which are conjugates of cell-binding antibodies and toxins, show considerable promise in the treatment of certain cancers. Genetic engineering is increasingly being used to refine and modify these conjugates, and it is now possible to design, express and purify completely recombinant therapeutic molecules.     

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes

 Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans. Received: 4 April 1996 / Accepted: 31 May 1996     

Movement of the tube cell in the lily style in the absence of the pollen grain and the spent pollen tube

Our model proposes that pollen tube growth is a form of cell movement where the tube tip can be considered analogous to a migrating cell which leaves a trail of extracellular matrix (the spent pollen tube) behind. We demonstrate that the tube cell can convey the sperm cells to the ovule and effect fertilization even in the absence of the pollen grain and the spent pollen tube. Adhesion is an integral part of cell attachment and movement in animal systems. We show that in vivo-grown pollen tubes grow beneath the cuticle of the stylar transmitting tract epidermis and directly adhere to one another and the outer wall of the epidermal cells. A fibrous wall material is found to cover the tip of the pollen tube cell wall and the surface of the transmitting tract cells where the two adhere. Fixation methods to preserve adhesive compounds were used. The pollen-tubes grown in vivo, but not in vitro, show star-shaped clusters of F-actin microfilaments in the region back from the tip, as seen by rhodamine-phalloidin staining. These configurations are similar to focal adhesions seen in moving animal cells.     

Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from L-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM L-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of L-threonine, glycine being the major product at low L-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20000 X g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous two-carbon compound detected when intact mitochondria catabolized L-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from L-threonine in rat liver mitochondria occurred primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.