UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region. Changes in organization of MTs in the irradiated region were analyzed by EM serial section analysis coupled with 3-D computer reconstruction. Metaphase cells irradiated 2 to 4 microns below the spindle pole (imaged by polarization optics) lost birefringence in the irradiated region. Peripheral spindle fibers, previously curved to focus on the pole, immediately splayed outwards when severed. We demonstrate via serial section analysis that following irradiation the lesion was devoid of MTs. Within 30 s to 1 min, recovery in live cells commenced as the severed spindle pole moved toward the metaphase plate closing the lesion. This movement was concomitant with the recovery of spindle birefringence and some of the severed fibers becoming refocused at the pole. Ultrastructurally we confirmed that this movement coincided with bridging of the lesion by MTs presumably growing from the pole. The non-irradiated half spindle also lost some birefringence and shortened until it resembled the recovered half spindle. Anaphase cells similarly irradiated did not show recovery of birefringence, and the pole remained disconnected from the remaining mitotic apparatus. Reconstructions of spindle structure confirmed that there were no MTs in the lesion which bridged the severed spindle pole with the remaining mitotic apparatus. These results suggest the existence of chromosome-to-pole spindle forces are dependent upon the existence of a MT continuum, and to a lesser extent to the loss of MT initiation capacity of the centrosome at the metaphase/anaphase transition.

Supraclavicular node biopsy in testicular tumors.

Fifty-seven patients with germinal cell carcinoma of the testis were evaluated routinely, with the addition of a supraclavicular node biopsy as a final staging procedure. Five patients showed more extensive disease with the addition of this staging modality.

Pelvic lymphadenectomy in stage A prostatic cancer.

Pelvic lymphadenectomy as a staging procedure in clinically apparent prostatic adenocarcinoma has long been recognized and its value appreciated. Twenty-three recent cases from the University of Colorado of clinically unapparent carcinoma of the prostate were studied with this modality, 5 Stage A1 and 18 Stage A2 tumors. Four of the 18 Stage A2 tumors but none of the A1 lesions after negative staging procedures revealed metastatic disease to the pelvic lymph nodes. Our experience indicated this modality should be employed in selected cases of incidental adenocarcinoma of the prostate.

Stromal hypocellularity and encapsulation in organ cultures of human prostate: application in epithelial cell isolation.

The primary objective of our study was to obtain pure cultures of prostatic epithelium. The phenomena of encapsulation by epithelial cells and hypocellularity in stroma occurred when explants of human prostatic tissue were maintained in suspension cultures. Hypocellularity progressed with time and was more pronounced in encapsulated explants. When encapsulated explants were allowed to attach to the substrate they formed an outgrowth of epithelial cells in a monolayer. The significance of these findings is in the use of the described changes in isolating and establishing epithelial cultures of human prostatic epithelium. These cultures are required for studies on the biology of prostatic epithelium and the etiology and treatment of prostatic neoplasia.

Ultrastructural changes in human prepubertal prostatic epithelium grown in vitro.

Ultrastructural changes in the prepubertal human prostatic epithelium maintained in vitro are described and are compared with the ultrastructure of the same tissue before culture. In cultured cells, rough endoplasmic reticulum and Golgi complex are poorly represented and the latter also loses its polarity. Increase in cytoplasmic microfilaments is discussed in relation to possible vitamin A deficiency. Primary and secondary lysosomes, arising from autophagy and endocytosis, occur in large numbers as autophagosomes, myelin figures, residual bodies, and multivesicular bodies. Prostatic acid phosphatase activity, an important secondary sex characteristic, is influenced by sex hormones and malignancy; since this enzyme is lysosome-associated, special emphasis is placed on lysosomal changes. Some ultrastructural changes in rough endoplasmic reticulum, Golgi complex, and the lysosomal system are similar to those observed after castration. This study presents ultrastructure of cultured cells which form the basis for studies involving neoplastic transformation, aging, and hormonal manipulation using an in vitro model. This is necessitated by the absence of an in vivo animal model for prostatic neoplasia; hence studies on prostatic oncogenesis, and age-related phenomenon, must be done on cells in vitro. Significance of this study is enhanced by the fact that normal human prepubertal prostate has not been studied before and normal viable prostate is generally not available for investigations.

The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex.

The DNA of Escherichia coli has been isolated in a compact structure containing small amounts of protein and RNA and having a sedimentation coefficient of approximately 3200 S. The molecular weight of the DNA in the complex is very large (probably higher than 10(9)); the protein is predominantly core RNA polymerase; the RNA is chiefly nascent messenger and ribosomal chains. Solutions containing the complex have low viscosities; this plus its sedimentation rate suggest that the DNA is in a tightly folded conformation. The DNA unfolds after exposure to RNase or heat; this indicates that an RNA component of the complex is involved in stabilizing the structure.

Isolation and identification of the human malignant prostatic epithelial cell in pure monolayer culture.

Isolation of the human malignant prostatic epithelial cell in pure monolayer culture was accomplished by clonal growth of single cells derived from metastatic deposits of prostatic carcinoma. Identification of these cells was established by the fact that the acid phosphatase of the prostatic epithelial cell is not inhibited by immersion in 10 per cent neutral formalin up to 24 hours, whereas all other acid phosphatases are destroyed by the formalin.

Identification of cultured, human, malignant, prostatic epithelial cells.

Identification of cloned cells is necessary for experimentation with them. This paper details a method for the identification of cultured human malignant prostatic epithelial cells derived from metastatic deposits of prostate cancer by localization of a specific rabbit antiserum to human prostatic acid phosphatase in the cells.

Bacterial stimulation and granulocyte inhibition of granulopoietic factor production.

We attempted to determine the effect of live bacteria (Staphylococcus epidermidis) on granulocyte colony-stimulating-factor production by human peripheral blood mononuclear cells (monocytes and lymphocytes) in vitro. Addition of bacteria to mononuclear-cell cultures enhanced colony-stimulating-factor production by these cells, as assayed on both human and mouse bone marrow. Addition of peripheral blood granulocytes to parallel cultures eliminated this enhancement effect, presumably by bacterial removal or inactivation. These data suggest that micro-organisms may have a pivotal role in granulocyte production and maturation by serving as a stimulus to increase colony-stimulating-factor production and also as negative control through their removal by the newly formed granulocytes.