Investigation of the attachment of bovine corneal endothelial cells to collagens and other components of the subendothelium : Role of fibronectin

Bovine corneal endothelial cells adhered equally well to a variety of collagens (types I, III, IV and V) consistent with a role for fibronectin in this process. They did not exhibit a preferential binding to collagen type IV—as might be anticipated if laminin were to play a significant role in their adhesion. Inhibition studies with anti-fibronectin antibodies demonstrated the importance of endogenous fibronectin in the mediation of attachment. Consistent with this, binding did not appear to require the presence of exogenous protein, since cells bound to collagens equally well in the presence or absence of added fibronectin and binding was not stimulated by pretreatment of collagens with this protein.     

Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.     

Pharmacokinetics of chlorogenic acid and rutin in larvae of Heliothis zea

Studies using [³H]chlorogenic acid and [³H]rutin demonstrated that the kinetics of uptake of these plant phenolics into the haemolymph of 5th-instar Heliothis zea (Boddie) following actue oral administration is a first-order process. The total quantity of either phenolic present in the haemolymph within 1 hr amounts to 5% or less of the total ingested dose. Based on TLC analyses, 80% or more of the radioactivity in the haemolymph occurs as the parent phenolic. Retention of [³H]-chlorogenic acid or [³H]-rutin in H. zea following chronic feeding from 1st to 3rd-instar larvae is also linearly related to dietary dose. Chlorogenic acid and rutin are both equitoxic and equivalent in bioavailability to H. zea.Loss of [³H]-rutin from the haemolymph of 5th-instar larvae following injection is biphasic. One half of the injected dose is excreted in the frass in the first 6 hr after injection; the other half is thereafter eliminated at 1/20th of the initial rate. Analyses of extracts of frass by thin-layer chromatography indicate that after either chronic or acute feeding 90% of the ingested phenolic is excreted unchanged. Possible sites and modes of action of phenolics in insects are discussed in light of these findings.     

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.     

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.     

EFFECT OF CYANIDE AND ELECTRICAL STIMULATION ON PHOSPHOINOSITIDE METABOLISM IN LOBSTER NERVES

The contents of phosphoinositides, ATP, glucose and lactate in leg and claw nerves of the lobster were determined. Nerves were also analysed after cyanide poisoning, after electrical stimulation, and 1 h after removing the leg from the lobster. Cyanide poisoning decreased the levels of ATP and glucose and increased the content of lactate but did not alter the levels of phosphoinositides. Nerves left in situ for 1 h after disconnection from the central nervous system exhibited a decrease in the content of tri-phosphoinositides (TPI) of 50 per cent, without changes in ATP, glucose or lactate. The TPI change was reversed after incubation for 1 h in oxygenated seawater. Nerves labelled in vivo with ³²P were removed and stimulated at 50 Hz for 5 min. The turnover of TPI phosphorus increased on stimulation in both normal and cyanide-poisoned nerves. In contrast, turnover of ATP increased after stimulation in normal nerves but not in cyanide-treated nerves. We sought to determine whether polyphosphoinositides play a greater role in resting metabolism of the nerve or in the conducting mechanisms. Our results make more likely the involvement of TPI in permeability changes of neural membranes during excitation.     

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide₆₅₀) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide₆₃₂) which is not. Dark-grown leaves placed in solutions of δ-aminolevulinic acid accumulate protochlorophyllide₆₃₂. Protochlorophyllide₆₅₀ and protochlorophyllide₆₃₂ can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide₆₅₀ in the dark, even in the presence of excess δ-aminolevulinic acid, except when a stockpile of protochlorophyllide₆₃₂ is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide₆₅₀ regeneration is accompanied by a decrease in protochlorophyllide₆₃₂. These experiments suggest that protochlorophyllide₆₃₂ may be converted to protochlorophyllide₆₅₀.