Lymphatic endothelial celline (CH3) from a recurrent retroperitoneal lymphangioma

Summary An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium. Supported in part by Arizona Disease Control Research Commission contracts 8277-000000-1-1-AT-6625 and ZB-7492. Presented in part at the 10th International Congress of Lymphology in Adelaide, Australia, August 1985.     

Genethics: project accountability via evaluation of teacher and student growth.

Accountability through demonstrated learning is increasingly being demanded by agencies funding science education projects. For example, the National Science Foundation requires evidence of the educational impact of programs designed to increase the scientific understanding and competencies of teachers and their students. The purpose of this paper is to share our human genetics educational experiences and accountability model with colleagues interested in serving the genetics educational needs of in-service secondary school science teachers and their students. Our accountability model is facilitated through (1) identifying the educational needs of the population of teachers to be served, (2) articulating goals and measurable objectives to meet these needs, and (3) then designing and implementing pretest/posttest questions to measure whether the objectives have been achieved. Comparison of entry and exit levels of performance on a 50-item test showed that teacher-participants learned a statistically significant amount of genetics content in our NSF-funded workshops. Teachers, in turn, administered a 25-item pretest/posttest to their secondary school students, and collective data from 121 classrooms across the United States revealed statistically significant increases in student knowledge of genetics content. Methods describing our attempts to evaluate teachers' use of pedagogical techniques and bioethical decision-making skills are briefly addressed.     

Antioxidants in sun and shade leaves of sour orange trees (Citrus aurantium) after long-term acclimation to elevated CO2

Antioxidative systems and the contents of pigments, malondialdehyde,soluble protein, and carbohydrate were investigated in sun-and shade-acclimated leaves of sour orange (Citrus aurantium)trees that had been grown for 7.5 years under ambient and elevated(+300 µmol mol^–1) atmospheric CO₂ concentrations.Sunacclimated leaves contained higher ascorbate, glutathioneand soluble carbohydrate contents and higher catalase activitiesthan shade-acclimated leaves. The activities of superoxide dismutases,which belonged to the family of Cu/Zn-isozymes, were similarin sunand shade-acclimated leaves and decreased in responseto enhanced CO₂. In shade-acclimated leaves, none of the otherparameters studied was affected by elevated CO₂. In sun-acclimatedleaves elevated CO₂ caused increases in carbohydrate and ascorbatecontents. There was no evidence for enhanced lipid peroxidationas assessed from the determination of the malondialdehyde contentsunder either conditions. Key words: Ascorbate, catalase, CO₂ enrichment, global change, glutathione, superoxide dismutase     

Plant growth analysis: an evaluation of experimental design and computational methods

Journal of Experimental Botany, Vol. 47, No. 302, pp. 1343–1351,September 1996 On page 1345, equation (2) should have read:      

Cell growth patterns and lens geometry: a quantitative study from three-dimensional reconstructions

The growth of the lens of the sea lamprey, Petromyzon marinus, was studied over the 5 years of larval development. Whole lenses (25) and Golgi-impregnated cells (393) were reconstructed with computer-assisted microscopy. Several cellular geometric parameters (length, width, curvature, surface, volume, shape) were correlated with the position of the cell's base on the lens capsular perimeter. Based on these correlations, the cells formed four groups that correspond to the central anterior, germinative, transitional and cortical fiber zones. A fifth zone, containing nuclear fiber cells, never stained. Lens growth is exponential during the 5 years. The anterior epithelium increases in size and in cell number by cell growth and division. The posterior mass increases in cell number by recruitment and increases in size by cell growth. A model is proposed to account for the size and shape of the lens based upon the coupling of anterior and posterior growth patterns. Four zonal boundaries are defined by changes in cell growth patterns. With growth, cells are subsumed into adjacent zones and zonal boundaries move away from the lens center. We find no support for the suggestion that cells migrate centrally.     

Collagen in the egg shell membranes of the hen

Collagen-like proteins have been found in the egg shell membranes of the hen. Materials similar to types I and V collagens were detected in each of the two layers of this membrane, the thick outer membrane and the thin inner membrane. Collagen was extracted by acid-pepsin digestion and isolated by differential salt precipitation. Identification of type-specific collagen-like material was established by coelectrophoresis on SDS-polyacrylamide gels using known collagen standards. These bands were susceptible to digestion by bacterial collagenase. From differential staining of the gels it was estimated that the ratio of collagen types I:V was approximately 100:1. Further confirmation of these biochemical results was obtained with immunofluorescence microscopy using type-specific antisera against chicken types I and V collagen with the indirect sandwich technique. Both the inner and outer shell membranes contained the two types of collagen. Within each membrane, the large, coarse 2.5-micron fibers contained predominantly type I collagen-like material, while type V collagen was mainly associated with the delicate narrower fibers of approximately 0.6-micron diameter. These tended to be concentrated in the inner membrane. At the electron microscopic level, both types of fibers were coated with glycoproteins that stained positively with ruthenium red. The deposition of these collagen-like substances by the hen oviduct on to the surface of the developing egg is an additional example of interstitial-type collagen synthesis and secretion by epithelial rather than by mesenchymal cells.     

Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson's Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best's carmine, Page-Green method for inclusion bodies, and Snook's reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.     

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

Inhibition of tumor cell invasion by verapamil.

Verapamil, a calcium channel antagonist, inhibits murine B16 melanoma and colon adenocarcinoma C26 tumor metastasis by altering platelet aggregation [Tsuruo, T., et al. (1985) Cancer Chemother. Pharmacol., 14:30-33]. However, the role of calcium homeostasis in regulating several biochemical pathways implicated in other steps of the metastatic cascade suggests that calcium channel antagonists could also inhibit metastasis by other mechanisms. In this report, non-toxic doses of verapamil reversibly decreased human A375M and C8161 melanoma cell invasion and metastasis in a dose-dependent manner. Verapamil reduced cellular invasion and metastases by up to 96% (range 78-96%). Concomitantly, verapamil disrupts microtubule and microfilament organization and inhibits unidirectional cell migration but does not affect cellular adhesion to endothelial monolayers or reconstituted basement membranes. In addition, tumor cells treated with verapamil have a decrease in mRNA of type IV collagenase, a proteinase important in tumor cell degradation of basement membranes. Collectively, these data offer additional evidence regarding the mechanisms of action of verapamil as an anti-metastatic agent.     

Head-tail connector of bacteriophage lambda

The head-tail connector of phage λ, a protein knob inside the head shell to which the tail attaches, is composed primarily of head protein gpB ⁴ and its cleaved form gpB¹. All of the gpB and gpB¹ in the virion is located in the connector. gpFII, the protein that is thought to form the site on the head to which the tail binds, is also located in the connector. Head proteins gpE, gpD, X1 and X2 are not components of the connector. These assignments were made by disrupting virions with guanidine hydrochloride, in such a way that heads and tails separate with the connectors attached to the tails, and determining which head proteins co-purify with the tails.We find that lysates from a λE^? infection contain a high proportion of tails with connectors attached. (Gene E codes for the major component of the head shell.) Connectors are also present on tails from a λE^?C^? infection, arguing that gpE, gpC, and their processed forms, X1 and X2, are all unnecessary for assembly of biologically competent connectors. The gpB in the connectors on E^? and E^?C^? tails is in the uncleaved form. Connectors are not seen on tails from infections by λE^?B^?, λE^?FII^?, or λE^? in a groE^? host.