Factors influencing phenotypic diversity of human prostate carcinoma cells metastasizing in athymic nude mice

Three related human prostate carcinoma cell lines, PC-3, 1-LN-PC-3-1A (1-LN), and 1-LN-PC-3-1A clone 4 (clone 4) were compared in terms of relative metastatic capacity in adult and young male nude mice. Only 1-LN produced lung metastases after intravenous injection into both 6- to 8-week-old and 4-week-old nude mice, as well as in mice injected intraperitoneally. The extent of the phenotypic diversity exhibited by these human prostate tumor lines was influenced by inherent dissemination ability, age of the host, and route of injection. These lines provide a useful system for the analysis of the biology of human tumor metastasis in nude mice.     

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Primary optic projections in the rabbit. Study using the horseradish peroxidase anterograde labeling technic

The primary visual pathways, in particular those to the lateral geniculate body, of 11 albino and 7 pigmented rabbits, were studied using the method of anterograde labelling with horseradish peroxidase following injection of the tracer into the vitreous body of one eye. A heavy projection to the contralateral dorsal lateral geniculate nucleus was seen in all animals. In both albino and pigmented animals a region devoid of label was present in the medial part of the alpha sector of the nucleus. This region corresponded to a compact, oval or wedge-shaped field of terminal label in the ipsilateral nucleus, which was much heavier in pigmented than in albino rabbits. In the ventral lateral geniculate nucleus, contralateral retinal input was almost entirely confined to the caudal half of the lateral sector of the nucleus, where two laminae of dense terminal label, separated by a less densely labelled area, were oriented parallel to one another and to the optic tract. This bilaminar distribution of retinal afferents to the ventral lateral geniculate nucleus has not been described in previous studies. The ipsilateral projection was to the dorsal part of the lateral sector and was most prominent in pigmented animals. The "intergeniculate leaflet" received a prominent contralateral input in all animals, and a clear ipsilateral input in pigmented animals, which overlapped with the contralateral input. Projections to other primary visual centres (pretectal nuclei, superior colliculus, nuclei of the accessory optic tract) are also described.     

A morphometric study on the effects of ethylene treatment in promoting abscission of tobacco flower pedicels

The ultrastructural changes observed in ethylene-induced abscission of tobacco flower pedicels (Nicotiana tabacum L. `Little Turkish') were studied by the techniques of morphometric analysis. The surface area of the membranes, relative volume of the organelles, and the number of organelles were determined for both ethylene-treated and control cells. In pedicels exposed to ethylene for 4.5 to 5 hours, abscission was evident within the separation zone. The most significant change in cell structure was observed in the surface area of the rough endoplasmic reticulum which more than doubled with ethylene treatment of the tissue.     

Localization of the carboxyl terminus of Band 3 to the cytoplasmic side of the erythrocyte membrane using antibodies raised against a synthetic peptide

Polyclonal antibodies were raised in rabbits against a synthetic peptide which corresponds to the 12-amino acid carboxyl-terminal sequence of murine erythrocyte Band 3. Immunoblots of ghost membrane proteins showed that the antibody specifically recognized murine or rat Band 3 but not human or canine Band 3. The antibody also bound to murine ghost membranes applied directly to nitrocellulose but not to human ghost membranes. This shows that the carboxyl terminus of Band 3 is available for antibody binding in ghost membranes and that the carboxyl-terminal sequences of human and mouse Band 3 are not identical. The specificity of the antibody for the carboxyl terminus of Band 3 was confirmed by the loss of antibody binding after digestion of detergent-solubilized ghost membrane proteins with carboxypeptidase Y. In addition, carboxyl-terminal fragments of Band 3 generated by protease treatment of cells or ghost membranes were positive on immunoblots while amino-terminal fragments were negative. In contrast, protease-treated stripped ghost membranes did not contain a carboxyl-terminal fragment of Band 3 that was detectable on immunoblots. The carboxyl terminus of Band 3 was localized to the cytoplasmic side of the erythrocyte membrane since antibody binding as determined by immunofluorescence occurred in ghosts and permeabilized cells but not in intact cells. In addition, competition studies using enzyme-linked immunosorbent assays and immunoblots showed that cells and resealed ghosts competed poorly for antibody compared to ghost membranes, inside-out vesicles, or albumin-conjugated peptide.     

Differential effects of epidermal growth factor and transforming growth factor-beta on synthesis of Mr = 35,000 surfactant-associated protein in fetal lung

Differentiation of pulmonary Type II epithelial cells in late gestation is associated with the synthesis of pulmonary surfactant required for adaptation to air breathing at birth. In the present work, induction of synthesis of a Type II epithelial cell protein, surfactant-associated glycoprotein of Mr = 35,000 (SAP-35) was studied in human fetal lung tissue obtained at 15-24 weeks of gestation. SAP-35 content increased during organ culture in the absence of exogenous hormones. Epidermal growth factor or triiodothyronine stimulated the induction of SAP-35 synthesis during culture. Stimulation by epidermal growth factor (EGF) was detected as early as 2 days and persisted for up to 5 days in culture. Response to EGF was dose-dependent (0.01-10 ng/ml) and was associated with enhanced incorporation of [35S]methionine into immunoprecipitable SAP-35. Increased SAP-35 synthesis was associated with increased SAP-35 RNA as assessed by Northern blot and hybridization assays with human SAP-35 cDNA. Effects of EGF were comparable to the induction of SAP-35 synthesis by 8-bromo-cAMP. In contrast to the stimulatory effect of EGF and triiodothyronine, SAP-35 content was inhibited by transforming growth factor-beta. Both the stimulatory and inhibitory effects of these agents on SAP-35 content were associated with concomitant changes in SAP-35 synthesis. These findings demonstrate multihormonal control of SAP-35 expression and strongly implicate both EGF and transforming growth factor-beta in the regulation of surfactant apoprotein synthesis.     

Potassium homeostasis in the nervous system of cephalopods and crustacea

1. Previous work has shown that nerve activity is associated with a significant release of potassium in the vicinity of the axonal membrane. Several mechanisms are normally present which reduce K+ accumulation in the extra-axonal space. 2. In intact connectives of the crayfish, Procambarus clarkii, repetitive stimulation of the giant axons was associated with an apparent hyperpolarization measured by an interstitial microelectrode, which most probably corresponds to depolarization of the inner face of the perineurial cells by K+ ions leaving the axons. 3. In desheathed connectives of the crayfish, potassium accumulated during long depolarizing voltage-clamp pulses but cleared away very quickly at the end of the pulse. 4. In the small squid, Alloteuthis subulata, repetitive stimulation of giant axons in situ in fresh and well-perfused animals did not result in a large decrease in the positive after potential (undershoot), reflecting the absence of potassium accumulation. A similar absence of accumulation was observed in vitro for carefully and freshly dissected isolated axons from live squids. 5. In both cases, deterioration of the physiological state of the axon was accompanied by a significant potassium accumulation. Potassium accumulation could also be reversibly enhanced by decreasing the osmotic pressure of the bathing medium, whereas hyperosmotic solutions had the opposite effect. These results are compatible with the idea that Schwann cells around the axon play a key role in K+ homeostasis. 6. Experiments on giant axons of the large squid species, Loligo forbesi confirmed the observations made on Alloteuthis in that fresh preparations exhibited little potassium accumulation. Under voltage-clamp conditions, 10 ms depolarizing pulses to various potential levels did not induce any accumulation in these preparations as reflected by the outward tail current. Large accumulation was observed in older axons under similar experimental conditions. 7. A large peri-axonal space associated with healthy glial cells appears to be a prerequisite for efficient K+ homeostasis in both crayfish and squid. Other mechanisms involving specific transport mechanisms across axonal and glial membranes are also likely to be involved.     

A third form of the G protein beta subunit. 2. Purification and biochemical properties.

Visual excitation in cones is thought to involve a cone-specific G protein (cone transducin) that transduces the light signal detected by the cone visual pigment into an increase in the enzymatic activity of a cGMP phosphodiesterase. In the preceding paper, we have shown that the G beta 3 isoform of G proteins is specifically localized in bovine cone photoreceptors and proposed that it might be a component of cone transducin. We reported here the purification from bovine retinal extract of a cone-specific T beta 3 gamma complex (where T is transducin), which is composed of a G beta 3 subunit and an immunochemically distinct G gamma subunit. Our purification of this complex is based on a two-stage procedure; the first stage consists of a series of column chromatographies that yield a mixture of purified T beta gamma substantially enriched in T beta 3 gamma, and the second stage involves the removal of all of the rod-specific T beta 1 gamma from the mixture using an affinity column of immobilized monoclonal antibodies directed against the rod T gamma subunit of transducin. Using this procedure, we were able to obtain sufficient amounts of T beta 1 gamma and T beta 3 gamma to begin a comparative study of their properties. We showed that T beta 3 gamma is distinguishable from T beta 1 gamma by isoelectric focusing under nondenaturing conditions. The G beta 3 polypeptide of T beta 3 gamma also migrates slightly slower than the G beta 1 polypeptide of T beta 1 gamma on denaturing polyacrylamide gels. Analysis of the interactions of T beta 3 gamma with other retinal proteins indicated that it has a lower affinity for the T alpha subunit of rod transducin but appears to complex with a phosducin-like protein. The differences in the intrinsic biochemical properties of T beta 3 gamma as compared to T beta 1 gamma may partially account for the lower light sensitivity of cones.     

Macrovascular and microvascular endothelium during long-term hypoxia: alterations in cell growth, monolayer permeability, and cell surface coagulant properties

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a approximately 3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro- and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.