Governing step of metastasis visualized in vitro

Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell’s basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547–552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085–12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis.     

Feature integration in pattern perception

The human visual system is able to effortlessly integrate local features to form our rich perception of patterns, despite the fact that visual information is discretely sampled by the retina and cortex. By using a novel perturbation technique, we show that the mechanisms by which features are integrated into coherent percepts are scale-invariant and nonlinear (phase and contrast polarity independent). They appear to operate by assigning position labels or “place tags” to each feature. Specifically, in the first series of experiments, we show that the positional tolerance of these place tags in foveal, and peripheral vision is about half the separation of the features, suggesting that the neural mechanisms that bind features into forms are quite robust to topographical jitter. In the second series of experiment, we asked how many stimulus samples are required for pattern identification by human and ideal observers. In human foveal vision, only about half the features are needed for reliable pattern interpolation. In this regard, human vision is quite efficient (ratio of ideal to real ≈ 0.75). Peripheral vision, on the other hand is rather inefficient, requiring more features, suggesting that the stimulus may be relatively underrepresented at the stage of feature integration.     

Equilibration rates in lipid monolayers

A theoretical analysis is given for the rate of change of domain sizes in lipid monolayers at the air–water interface. The calculation is applicable to liquid domains formed from binary mixtures of lipids that form two coexisting liquid phases. Under conditions where the two lipid molecules have approximately equal areas, the equilibration rate does not involve macroscopic hydrodynamic flow in the subphase but rather depends on the diffusion coefficient of the lipid molecules. The calculation shows that the equilibration rate in binary mixtures of cholesterol and phosphatidylcholine is remarkably slow, the radius of a typical 20-μm diameter domain changing by as little as a part in a million per second. Under these circumstances, equilibration times of the order of days or weeks are expected. Even with such long times, the final state reached by the monolayer will in general be a state of metastable equilibrium, rather than true equilibrium.     

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.     

Very short-term plasticity in hippocampal synapses

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2–10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca²⁺ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.     

Assortative fertilization in Drosophila

The concept of gametic isolation has its origins in the 1937 edition of T. Dobzhansky’s Genetics and the Origin of Species. Involving either positive assortative fertilization (as opposed to self-incompatibility) or negative assortative fertilization, it occurs after mating but prior to fertilization. Gametic isolation is generally subsumed under either prezygotic or postmating isolation and thus has not been the subject of extensive investigation. Examples of assortative fertilization in Drosophila are reviewed and compared with those of other organisms. Potential mechanisms leading to assortative fertilization are discussed, as are their evolutionary implications.     

Proteins unfold in steps

I present results from an experiment on the dynamics of folding of a globular protein (bovine serum albumin). Employing a micro-mechanical technique, I perform the measurements on very few molecules (1–100). I observed a sequence of steps in time for both unfolding and refolding. The overall characteristic time of the process is thus built up of waiting times between successive steps. The pattern of steps is reproducible, demonstrating the existence of deterministic pathways for folding and unfolding. Certain symmetries in the patterns of steps may reflect the architecture of the protein’s structure.     

Retinoic acid alters photoreceptor development in vivo

Application of exogenous retinoic acid (RA) to zebrafish during the initial stages of photoreceptor differentiation results in a precocious development of rod photoreceptors and an inhibition of cone photoreceptor maturation. The acceleration of rod differentiation is observed initially within the ventral retina 3 days after fertilization, following 24 hr of RA application, and within the dorsal retina 4 days after fertilization, following 48 hr of RA application. The differentiation of rods was impeded significantly when the synthesis of endogenous retinoic acid was inhibited by citral prior to the initial stage of rod differentiation. RA-treated embryos labeled for bromodeoxyuridine (BrdU) uptake revealed that RA exerts its effect on a postmitotic cell population within the developing retina. During normal development in zebrafish, rod differentiation is most robust within the ventral retina, a region previously shown to be rich in RA. Our data suggest that the RA signaling pathway is involved in the differentiation and maturation of both the rod and cone photoreceptors within the developing zebrafish retina.     

Symmetry arguments in chemistry

The use (and misuse) of symmetry arguments in constructing molecular models and in the interpretation of experimental observations bearing on molecular structure (spectroscopy, diffraction, etc.) is discussed. Examples include the development of point groups and space groups for describing the external and internal symmetry of crystals, the derivation of molecular symmetry by counting isomers (the benzene structure), molecular chirality, the connection between macroscopic and molecular chirality, pseudorotation, the symmetry group of nonrigid molecules, and the use of orbital symmetry arguments in discussing aspects of chemical reactivity.     

Role of leptin in hypothalamic–pituitary function

A defect in the structure of the obese gene is responsible for development of obesity in the ob/ob mouse. The product of expression of the gene is the protein hormone leptin. Leptin causes weight loss in ob/ob and normal mice, it is secreted by adipocytes, and it is an important controller of the size of fat stores by inhibiting appetite. The ob/ob mouse is infertile and has a pattern of gonadotropin secretion similar to that of prepubertal animals. Consequently, we hypothesized that leptin might play a role in the control of gonadotropin secretion and initiated studies on its possible acute effects on hypothalamic–pituitary function. After a preincubation period, hemi-anterior pituitaries of adult male rats were incubated with leptin for 3 hr. Leptin produced a dose-related increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release, which reached peaks with 10⁻⁹ and 10⁻¹¹ M leptin, respectively. Gonadotropin release decreased at higher concentrations of leptin to values indistinguishable from that of control pituitaries. On the other hand, prolactin secretion was greatly increased in a dose-related manner but only with leptin concentrations (10⁻⁷–10⁻⁵ M). Incubation with leptin of median eminence–arcuate nuclear explants from the same animals produced significant increases in LH-releasing hormone (LHRH) release only at the lowest concentrations tested (10⁻¹²–10⁻¹⁰ M). As the leptin concentration was increased, LHRH release decreased and was significantly less than control release at the highest concentration tested (10⁻⁶ M). To determine if leptin can also release gonadotropins in vivo, ovariectomized females bearing implanted third ventricle cannulae were injected with 10 μg of estradiol benzoate s.c., followed 72 hr later by microinjection into the third ventricle of leptin (0.6 nmol in 5 μl) or an equal volume of diluent. There was a highly significant increase in plasma LH, which peaked 10–50 min after injection of leptin. Leptin had no effect on plasma FSH concentrations, and the diluent had no effect on either plasma FSH or LH. Thus, leptin at very low concentrations stimulated LHRH release from hypothalamic explants and FSH and LH release from anterior pituitaries of adult male rats in vitro and released LH, but not FSH, in vivo. The results indicate that leptin plays an important role in controlling gonadotropin secretion by stimulatory hypothalamic and pituitary actions.     

Localized gene action controlling intestinal neoplasia in mice

Mice heterozygous for the Apc^Min (Min) mutation develop adenomas throughout the intestinal tract. Apc is believed to be involved in cell migration, adhesion, and polarity. Adenoma multiplicity and growth rate are modulated by an unlinked modifier locus, Mom1. The secretory phospholipase Pla2g2a is a candidate for Mom1. Here, we investigate the range of action of Apc and Mom1. Analysis of chimeric Min mice indicates that the actions of both Apc and Mom1 are localized within the cell lineage that gives rise to intestinal tumors.     

Structural protein 4.1 is located in mammalian centrosomes

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.     

Probing the role of packing specificity in protein design

By using a protein-design algorithm that quantitatively considers side-chain packing, the effect of specific steric constraints on protein design was assessed in the core of the streptococcal protein G β1 domain. The strength of packing constraints used in the design was varied, resulting in core sequences that reflected differing amounts of packing specificity. The structural flexibility and stability of several of the designed proteins were experimentally determined and showed a trend from well-ordered to highly mobile structures as the degree of packing specificity in the design decreased. This trend both demonstrates that the inclusion of specific packing interactions is necessary for the design of native-like proteins and defines a useful range of packing specificity for the design algorithm. In addition, an analysis of the modeled protein structures suggested that penalizing for exposed hydrophobic surface area can improve design performance.     

Correct protein folding in glycerol

Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding/reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.     

Antigen presentation in retroviral vector-mediated gene transfer in vivo

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.     

Tissue engineering of cartilage in space

Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10⁻⁴–10⁻⁶ g) or Earth (1 g). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.