An improved rapid assay of glycogen synthase

As an alternative to rapid filtration washing the glycogen free of any unreacted UDP-[¹⁴C]-glucose by ascending chromatography (ethanol:water, 2:1) can be used. This technique also makes the filter paper assay of glycogen synthase much faster: The samples are ready for liquid scintillation counting in 30 min. Among the other advantages offered by this procedure, we should also mention that blanks are very low, large volumes of ethanol can be saved, and the unreacted UDP-[¹⁴C]glucose can be recovered by elution and recycled (it migrates with the front of the solvent).     

An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [³H]thymidine and [¹⁴C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.     

Degradation and covalent cross-linking of glutathione reductase by hemin

Hemin (ferriprotoporphyrin IX-chloride) can mediate the covalent cross-linking and degradation of yeast glutathione reductase. This reaction requires both NADPH and oxygen suggesting the involvement of a reduced oxygen species in the cross-linking and degradation process. During the course of the reaction the enzymatic activity of glutathione reductase is rapidly destroyed. Implications of these findings for a regulatory role of hemin in cell biology are discussed.     

Conformational stabilization of enzymes in covalent catalysis.

The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pH-induced structural transitions to inert forms that occur in the free enzyme species and other forms not covalently substituted.     

Bioautography of cell attachment proteins.

A simple method, termed bioautography, is described which permits the visualization of bands of biologically active collagen-dependent cell attachment protein (c-CAP) after gel electrophoresis. The principle of bioautography depends on the “staining” of electrophoretically separated c-CAP's by live mammalian cells. Bioautography demonstrates (a) two bands of cell attachment protein (c-CAP) in serum; and (b) electrophoretic mobility differences between c-CAP's derived from the sera of various mammalian species.     

Secondary-site attachment of coliphage lambda near the thr operon

Phage λ has been integrated at a secondary attachment site near the thr oporon of Escherichia coli. The integration caused a pleiotropic effect since both thrA and thrB enzyme activities were lost. Lysogenic, thr⁺ revertants regained thrA and thrB activities although the enzymes were synthetized constitutively at low levels. Four classes of prophage deletion strains were isolated with deletions extending into trpR and the thr operon. Genetic and biochemical analyses indicated a gene order: trpR .. λ .. thrA .. B .. C. Defective transducing phage carrying thr A, B,C were also isolated.     

A microquantitative method to characterize lectin-induced cytoagglutination

A microquantitative method to characterize lectin-induced cytoagglutination reactions is described. The assay, which utilizes an electronic particle counter, measures the disappearance of single cells from cell suspensions containing various concentrations of lectin. The method is sensitive, requiring only μg quantities of lectin, reproducible, and amenable to thorough statistical analysis. It was also utilized to quantitate the inhibition of lectin-induced cytoagglutination by various saccharides. The method was employed to characterize ConA-induced agglutination of guinea pig erythrocytes and Novikoff ascites hepatoma cells, and to investigate several parameters which influence lectin-induced cytoagglutination reactions.     

Concanavalin A mediated attachment and ingestion of red blood cells by macrophages.

The interaction of red blood cells and macrophages mediated by Concanavalin A (ConA) was studied using mouse peritoneal macrophages and fresh, homologous red cells. Erythrocytes exposed to ConA at 0.5 μg/ml, a condition that leads to a saturation of 3% of the ConA sites, were bound by macrophages at 22 °C. The ConA inhibitor, α-methylmannoside, prevented this attachment of red cells and largely reversed it when added to preformed macrophage-red cell rosettes up to 90 min. However, red cell attachment was essentially irreversible by 3 h. Electron microscopy showed a progressive increase in the degree of contiguity between red cells and macrophages with time, some macrophage projections distorting and partially encircling red cells at 3 h. Macrophages pretreated with high concentrations of ConA (25 μg/ml) also bound red cells. However, phagocytosis of adherent red cells did not occur at either 22 or 37 °C, even when both red cells and macrophages were pretreated with ConA. In contrast, phagocytosis of attached red cells was observed when preformed rosettes were exposed to ConA at a concentration of 5 μg/ml, and it was complete with ConA at a concentration of 25 μg/ml. These studies demonstrate that ConA in low concentration on red cells is detected by macrophages which form a progressively tighter bond with the red cell surface. However, it appears that phagocytosis can occur only under conditions in which a high density of ConA is established on the surface of the red cell.     

An enzymatic microassay for lactose

An enzymatic microassay for lactose using a lactase enzyme derived from Saccharomyces fragilis is described. The assay uses 50-μl samples, provides 100% hydrolysis of lactose, and is sensitive within the range of 12.5–500 nmol per sample. The assay has been validated against an assay for ¹⁴C lactose which involves thin-layer chromatographic isolation of lactose. The assay is sufficiently sensitive for use in physiologic studies.     

Evidence for the identification of lymphocyte activating factor as the adherent cell-derived mediator responsible for enhanced antibody synthesis by nude mouse spleen cells

Human adherent peripheral blood leukocytes spontaneously elaborate both a thymocyte proliferative factor and a factor which augments the in vitro anti-sheep erythrocyte (SRC) plaque-forming cell (PFC) response of nu/nu mouse spleen cells. Nonadherent leukocytes do not spontaneously elaborate either factor. The adherent cell-derived factors appear to have an identical molecular weight (approximately 14,500 Daltons) as determined by Sephadex gel filtration. The data support the hypothesis that the molecule(s) mediating both enhancing activities is identical to the previously described adherent leukocyte product, LAF.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated “intact” rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na⁺, ATP, glucose-6-P, glycerol formation and ⁴⁵Ca uptake and loss. Basal sugar transport was inhibited by low (10⁻⁸−10⁻⁵ M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10⁻⁴−10⁻³ M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca²⁺. These results suggest interaction with two different classes of adrenergic receptors, possibly of β₁ and β₂ types. Both low and high concentrations increased Na⁺ and K⁺ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na⁺ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na⁺ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of ⁴⁵Ca was decreased by low and increased by high adrenalin; ⁴⁵Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca²⁺ on sugar transport in muscle, with adrenalin affecting Ca²⁺ fluxes and distribution both directly and indirectly.     

A method for determining DNA and chondrocyte content of articular cartilage

A novel and precise method was devised to study the DNA and chondrocyte content of articular cartilage. It involved the sequential digestion of cartilage matrix with hyaluronidase, trypsin, and collagenase to release the chondrocytes. A direct cell count and DNA assays were then performed on the cells. The concentration of cells was the quotient of the total number of cells and the weight of cartilage used. The DNA content of cartilage is identical to the amount of DNA in the chondrocytes. Our data also confirmed the earlier findings that cell density and DNA content of articular cartilage decreased gradually to a relatively constant level as animals matured to adulthood.     

An experimental approach to the study of factors affecting algal colonization in a sublittoral kelp forest

Factors influencing the early colonization by benthic macroalgae and the early successional sequence in a subtidal Ecklonia radiata (C. Agardh) J. Agardh forest in Port Jackson (Sydney) were investigated using controlled experiments involving the microscopic sampling of settlement plates. The preliminary results indicate that the Tolerance model of succession may be applicable. The effect of grazers, and competition between different algal types were important and could, later in the succession, lead to the operation of the mechanisms suggested by the Facilitation or Inhibition models. Controls for the use of settlement plates showed that thick settlement plates led to an over-estimation of initial algal growth compared with the growth on natural patches of substratum. Other results suggested that thick plates excluded grazers. Borders of plates were investigated, but the establishment of algae was random over the plates′ surfaces and border effects were the result of physical or biotic factors acting on algae after settlement. An experiment was developed for use in subtidal areas to test for artifacts due to cages. It was found that the presence of the cages influences the community by attracting small snails. Fish were excluded from some areas to monitor their effects on initial algal growth. Exclusion of fish was found to cause a reduction in algal growth as well as increases in numbers of small invertebrates such as amphipods and copepods. The results suggested that the amount of algae initially present was affected by invertebrate grazers, the abundances of which were, in turn, affected by predation. The species diversity of the early algal growth did not show any consistent trends between high and low grazing pressure. Grazing was important in determining the temporal variability of early growth of individual species. Siltation was positively correlated with early algal growth; a mechanism to account for this effect is suggested. The application of the methods used in this study to future experimental studies of sublittoral community dynamics is considered.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

Stimulation of ion release from liposomes by the polyene antibiotic, filipin.

The effect of the polyene antibiotic, filipin, upon release of the ions Ca²⁺, Sr²⁺, SO₄^2? and phosphate out of phospholipid and phospholipid-cholesterol liposomal vesicles was studied. The addition of filipin at concentrations stoichiometrically comparable to the cholesterol concentration in the liposomes, resulted in 2–10 × stimulation of the rate of release of all of these ions. The filipin mediated stimulation of release of ions from liposomes was dependent upon the presence of cholesterol. The relative effectiveness of filipin increased when the mole percent of cholesterol incorporated into the liposomes increased from 10 to 50% and when the molar filipin:cholesterol ratio increased from 0.2 to 1.0. It has been previously shown that there is a 1:1 stoichiometry of interaction between filipin and cholesterol [Biochem. Biophys. Acta339, 57 (1974)]. The present studies suggest that this 1:1 stoichiometric interaction may also be responsible for the increased release of entrapped ions.A possible mechanism of action of polyene antibiotics is discussed which suggests that the rearrangement of membrane constituents occurring upon interaction of filipin with cholesterol is the basis for the enhancement of ion release. This would imply that the ion specificity observed upon interaction of polyene antibiotics with membranes would not only be determined by the polyene antibiotic itself, but also by the intrinsic properties of the membrane.     

A method for fractionating rat liver cell nuclei on equilibrium density gradients of CS2SO4.

Sonically disrupted nuclei from proliferating liver cells were fractionated in Cs₂SO₄ equilibrium density gradients. Nuclear constituents were concentrated in three bands designated as light band (LB, 1.21 g/cm³), middle band (MB, 1.26 g/cm³), and heavy band (HB, 1.32 g/cm³). Analysis of protein and nucleic acid distribution in gradients suggests preservation of some macromolecular interactions. Studies comparing distributions of radioactively labeled DNA after 1- or 120-min intervals following tritiated thymidine injection indicate enrichment of nascent DNA in LB and MB. This enrichment is sensitive to time and pressure of sonication. Furthermore, DNA-polymerase activity was demonstrated in the gradient fractions following removal of Cs₂SO₄, with most activity once again in the LB and MB. These results suggest this procedure as an initial step in the isolation of an enzymatically active DNA replication complex.