Vascular endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vessel wall. The aim of this study was to determine the distribution and identity of the gap junction structural proteins (connexins) expressed by endothelial cells in situ. Connexin expression in different regions of the rat aortic endothelium was analyzed with the use of indirect immunofluorescence microscopy and Western blotting. Connexin40 and connexin37 were present in most, if not all, of the thoracic and abdominal aortic endothelia in the form of maculae at cell-cell appositions. In contrast, connexin43 was undetectable in most endothelia but extremely abundant in small numbers of cells localized at the downstream edge of the ostia of branching vessels and at flow dividers, regions that experience turbulent shear stress from disturbed blood flow. To examine the relationship of shear stress and connexin43 expression, localized stress was induced by surgical coarctation of the aorta, which was sufficient to cause striking local upregulation of connexin43 within 8 days. Thus, increases in connexin43 levels are an endothelial response to mechanical stress.

A B S T R A C T Peripheral blood lymphocytes and splenocytes of patients with autoimmune disease were used to prepare human-human hybridomas that produce autoantibodies. Because exogenous immunization was not used, the hybridoma antibodies were derived from B cells that spontaneously produced autoantibodies. 108 hybrids grew from 4,254 wells (2.5%). Optimal conditions for obtaining hybridomas with the GM 4672 myeloma line included initial growth in 2-ml wells, the use of 44% polyethylene glycol, a mononuclear cell/GM 4672 cell ratio of 5:1, and prior stimulation of the B lymphocytes with pokeweed mitogen. Hybridoma supernatants had activity against ssDNA, platelets, and erythrocytes. The results demonstrate the feasibility of producing human-human hybridomas from lymphocytes of patients with various autoimmune diseases.

We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.

No abstract

Studies were performed with cellulosic filters and standard culture plates to compare methods of cell culture and differentiation of the cell line PC12, a clone originating from a rat pheochromocytoma. PC12 cells respond to nerve growth factor (NGF) by flattening of the cell body and subsequent extension of neurite-like processes. When PC12 cells are cultured in dishes without NGF, they have a diameter of approximately 3 to 7 micron and exhibit short processes of no longer than 3 to 5 micron. If PC12 cells are grown on a cellulosic filter they have the same average soma diameter and similar short processes extending laterally, but in addition have branching processes which extend as far as 10 to 15 micron into the filter substrate. When dish-cultured and filter-cultured cells are incubated with 50 ng/ml NGF they both exhibit differentiation-specific ultrastructural changes by 3 d of treatment. In the case of dish-cultured cells, large cytoplasmic processes exhibit an increase in the number of chromaffin cell-like secretory granules by 3 d of treatment. This characteristic is also demonstrated by filter-cultured cells, but the processes containing these granules are found concentrated within the cellulosic meshwork. Thus the timing of the NGF-elicited differentiation program is similar to both filter-cultured and dish-cultured cells, but the ultrastructural consequences are different. The filter-cultured PC12 cells exhibit a polarity not demonstrated by dish-cultured cells. Growing PC12 cells on cellulosic filters is a technique useful for "anchoring" neurons without the complication of the addition of extracellular matrix components. Filter-culture may represent a more in vivo-like method for studying neuronal growth and differentiation.