A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

The effect of ethanol on arachidonic acid metabolism in the murine peritoneal macrophage

Exposure to ethanol in man has been linked to an alteration of the immune surveillance system and reduced ability of the macrophage to undergo phagocytosis. Since ethanol has been suggested to alter membrane function and inhibit the production of calcium ionophore stimulated synthesis of prostaglandins and leukotrienes by the human neutrophil and transformed murine mast cell, the dose response effect of ethanol on the biosynthesis of icosanoids by the peritoneal macrophage during zymosan phagocytosis was studied. Peritoneal macrophages from two inbred strains of mice derived from a common stock (HS) and selected for sensitivity to ethanol (shoprt sleep [SS]/long sleep [LS]) were studies. Zymosan phagocytosis was found to lead to synthesis of LTC₄ (70 ng/10⁶ cells), 6-keto-PGF_1a (5 ng/10⁶ (3 ng/10⁶ cells). For the HS macrophage, ethanol caused a dose dependent inhibition of these lipid mediators as well as inhibition of phagocytosis and release of beta-hexosaminidase. However, a difference was observed in arachidonate metabolism stimulated by phagocytosis between the LS and SS mice below 100 mM ethanol. The SS mouse had a 50% inhibition of cyclooxygenase products at 86 mM ethanol with no inhibition of lipoxygenase metabolites. The LS mice had a trend suggesting increased lipoxygenase metabolites below 100 mM ethanol. At these levels of ethanol which can be found in man, these results suggest there may be differential production of lipid mediators under genetic control.     

Mechanisms of airway hypercontractility in basenji-greyhound dogs

The comparative effects of contractile agonists and physiological stimulation of the tracheal and bronchial smooth muscle (BSM) response were studied isometrically in situ in five Basenji-greyhound (BG) and six mongrel dogs. Frequency-response curves generated by bilateral stimulation of the vagus nerves (0-20 Hz, 15-20 V, 2-ms duration) elicited greater maximal contraction in mongrel trachea (36.8 +/- 8.1 vs. 26.9 +/- 4.0 g/cm; P less than 0.02) and exhibited greater responsiveness in mongrel BSM (half-maximal response to electrical stimulation 3.0 +/- 1.1 vs. 7.0 +/- 0.5 Hz; P less than 0.05) compared with BG dogs. However, muscarinic sensitivity to intravenous methacholine (MCh) was substantially greater in BG dogs; MCh caused contraction greater than 1.5 g/cm at a mean dose of 3.0 X 10(-10) mol/kg for BG dogs compared with 5.1 X 10(-9) mol/kg for mongrel controls (P less than 0.03, Mann-Whitney rank-sum test). In contrast to the muscarinic response, the contractile response elicited by intravenous norepinephrine after beta-adrenergic blockade was similar in trachea and bronchus for both mongrel and BG dogs. Our data confirm previous in vitro demonstration of tracheal hyporesponsiveness in BG dogs and demonstrate that the contraction resulting from efferent parasympathetic stimulation is less in the BG than mongrel dogs. However, postsynaptic muscarinic responsiveness of BG BSM is substantially increased. We conclude that a component of airway responsiveness in BG dogs depends directly on contractile forces generated postsynaptically that are nongeometry dependent, postjunctional, and agonist specific.     

Association between transfusion of whole blood and recurrence of cancer.

Transfusion affects the immune response to renal transplantation and may be associated with recurrence of various human neoplasms. Data from patients with colonic, rectal, cervical, and prostate tumours showed an association between transfusion of any amount of whole blood or larger amounts of red blood cells at the time of surgery and later recurrence of cancer. Recipients of one unit of whole blood had a significantly higher incidence of recurrence (45%) than recipients of a single unit of red cells (12%) (p = 0.03). Recipients of two units of whole blood also had a higher rate of recurrence (52%) than those receiving two units of red cells (23%) (p = 0.03). Recipients of any amount of whole blood had similar recurrence rates (38-52%). Recipients of four or more units of red blood cells had a higher rate of recurrence (55%) than those receiving three or fewer units of red blood cells (20%) (p = 0.005). Mortality due to cancer in patients receiving three or fewer units of red blood cells (2%) was similar to that in patients who did not have transfusions (7%) and significantly lower than that observed in patients receiving three or fewer units of whole blood (20%) (p = 0.003). A proportional hazards risk analysis showed that transfusion of any whole blood or more than three units of red blood cells was significantly associated with earlier recurrence and death due to cancer. These data support an association between transfusion and recurrence of cancer. They also suggest that some factor present in greater amounts in whole blood, such as plasma, may contribute to the increased risk of recurrence in patients who have undergone transfusion. Until the questions raised by retrospective studies of cancer recurrence and transfusion can be answered by prospective interventional trials with washed red blood cells, red blood cells should be transfused to patients with cancer in preference to whole blood when clinically feasible.     

Precocious induction of luteal activation and termination of delayed implantation in mink with the dopamine antagonist pimozide

The effects of the dopamine antagonist pimozide on the preimplantation delay phase of mink gestation were investigated in field and laboratory trials. Three doses of 0.1 mg pimozide in acetic acid administered on the 7th, 9th and 11th days after mating abbreviated gestation in Pastel kit female mink to a mean (+/- SEM) of 45.5 +/- 0.5 days, 10 days less than that observed in mink treated with vehicle only (55.6 +/- 0.6 days). In laboratory trials, four doses of 0.1 mg pimozide on the 7th, 9th, 11th and 13th day after mating resulted in embryo implantation at a mean of 25 +/- 4.3 days after mating while vehicle-treated control animals had mean preimplantation delay of 37 +/- 3.1 days. Luteal activation in the pimozide-treated group, as indicated by a rapid increase in circulating progesterone, began within 2 days after the first pimozide injection. No increase was observed in vehicle-treated mink until 6 or more days after the initiation of injections or 13 days after mating. It was concluded that pimozide, presumably by permitting endogenous secretion of prolactin, can induce precocious luteal activation and embryo implantation in the mink.     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.     

Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides

beta-Tubulin subunits isolated from chicken brain tissue and erythrocytes are distinguishable as unique biochemical species by electrophoretic and peptide mapping procedures. 1) The subunits of beta-tubulin exhibit major differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels that vary according to the pH and ionic strength of the gel. 2) The isoelectric points of urea-denatured beta subunits from brain tissue and erythrocytes are pH 5.1 and 5.4, respectively, whereas those of both alpha subunits are approximately pH 5.2.3) Two-dimensional peptide maps prepared with alpha-chymotrypsin or V8 protease show that alpha-tubulin peptides are indistinguishable, whereas beta-tubulin peptides are very different. Only one-third of the 15 major tyrosine-containing beta-tubulin peptides prepared with alpha-chymotrypsin are common to both beta-tubulin species. The data indicate that the beta-tubulin subunits of brain tissue and erythrocytes are biochemically distinct and may be different gene products. The presence of tubulin variants in brain tissue and erythrocytes may indicate special requirements for microtubule assembly and function in different cell types.