Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Physiological and Biochemical Mechanisms Involved in the Response to Abscisic Acid in Maize Coleoptiles

The role of abscisic acid (ABA) in controlling growth and developmenthas been studied in maize (Zea mays L.) coleoptile segments.Application of ABA reduces the elongation rate by about 50%and affects ion fluxes. In particular, proton extrusion is decreasedwhile potassium efflux is greatly enhanced. Apparently, ABAdoes not: seem to influence calcium influx from the apoplastinto the cytosol, but more likely it influences its efflux.Alteration of cytosolic calcium concentration may also be obtainedby increasing its release from internal stores. This possibilitymight be sustained by the increased hydrolysis of phosphatidylinositolupon ABA application. Change in the balance of ion fluxes shouldresult from regulation of transport mechanisms at the membranelevel and should produce changes in the transmembrane electricalpotential. The H⁺- ATPase and the ATP-dependent calcium transportactivities are both influenced by the treatment with ABA, –55%and –40%, respectively. Under these conditions [Ca²⁺]_cytand pH_cyt can be modified and, as a consequence of their regulation,they may play an important role in mediating the physiologicaland biochemical effects of ABA, acting as second intracellularmessengers. ¹Research supported by National Research Council of Italy, SpecialProject RAISA, Sub-Project N. 2, Paper n. 2782.     

Fc gamma RI and Fc gamma RII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells

As part of an effort to define the cytotoxic trigger molecules on human myeloid cells, the ability of the different Fc receptors for IgG (Fc gamma R) to mediate killing of tumor cell lines by monocytes and granulocytes was examined. This was accomplished by studying cytolysis of hybridoma cell (HC) targets bearing surface antibody directed toward the different Fc gamma R. The HC line, HC IV.3A, which bears Ig directed to the low affinity Fc gamma R (Fc gamma RII) on monocytes and neutrophils was lysed by human monocytes. The extent of lysis of HC IV.3A was approximately equal to that of anti-Fc gamma RI (the high affinity Fc gamma R on human monocytes) bearing HC lines (HC 32.2A and HC 62A) and was not augmented by treatment of the monocytes with interferon-gamma (IFN-gamma). In contrast, neutrophils lysed HC IV.3A and HC 32.2A only after activation with IFN-gamma. Since Fc gamma RI is not detectable on untreated neutrophils and is induced by IFN-gamma on these cells, lysis of HC 32.2A by IFN-gamma-activated neutrophils correlated with receptor induction. On the other hand, Fc gamma RII was present at equal levels on untreated and IFN-gamma-treated neutrophils, but only IFN-gamma-treated neutrophils mediated cytotoxicity via Fc gamma RII. In this case, enhanced killing appeared to be due to events other than an increase in Fc gamma RII number. Neither untreated nor IFN-gamma-treated neutrophils mediated the lysis of the anti-Fc gamma RIII bearing HC 3G8A. Thus, binding to the tumor target via this Fc receptor does not lead to lysis and may initiate signals distinct from those triggered through Fc gamma RI or Fc gamma RII. Surprisingly, HC bearing high amounts of mouse IgG1 antibody of irrelevant specificity were also lysed by monocytes. This lysis was blocked by soluble IV.3 antibody and thus appeared to be due to binding of the Fc portion of the surface Ig to Fc gamma RII on monocytes. Furthermore, monocytes from donors with a form of Fc gamma RII incapable of binding aggregated mouse IgG1 did not lyse these HC, but displayed normal lysis of HC IV.3, demonstrating that this structurally different Fc gamma RII remained a functional trigger molecule. Overall, these studies have demonstrated the specificity of Fc receptors in triggering monocyte- and granulocyte-mediated antibody-dependent tumor cell killing and have begun to dissect functional similarities and differences among the three defined Fc gamma R on human myeloid cells.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Fat cell beta-adrenergic receptor in the hypothyroid rat. Impaired interaction with the stimulatory regulatory component of adenylate cyclase

Fat cells from the hypothyroid rat fail to synthesize cyclic AMP in response to beta-adrenergic agonists, although possessing normal amounts of beta-adrenergic receptors (R) and catalytic adenylate cyclase activity. Membranes of hypothyroid rat fat cells contain Mr = 42,000 (major form), 46,0000, and 48,000 (minor forms) peptides of the stimulatory guanine nucleotide-binding regulatory component (Ns) radiolabeled in the presence of cholera toxin and [32P]NAD+. Maps of fragments generated by partial proteolysis of these radiolabeled peptides are virtually identical in hypothyroid and euthyroid preparations. Two-dimensional gel electrophoresis showed that the size and charge of the Mr = 42,000, 46,000, and 48,000 radiolabeled peptides are similar in euthyroid and hypothyroid rat fat cell membranes. Extracts of hypothyroid rat fat cell membranes express normal amounts of Ns activity as measured by their ability to reconstitute the adenylate cyclase of membranes of S49 mouse lymphoma cyc- mutant cells which lack functional Ns activity. Hybridization of hypothyroid rat fat cells with donor membranes of normal rat fat cells, rat hepatocytes, or S49 cyc- cells restores the beta-adrenergic response of these fat cells. Pretreating the donor membranes with a beta-adrenergic antagonist covalent label blocks the ability of these membranes to restore the response of the cells. Rat hepatocytes pretreated with a beta-adrenergic antagonist covalent label do not accumulate cyclic AMP in response to isoproterenol. Hybridization of these receptor-deficient hepatocytes with fat cell ghosts of euthyroid rats restores beta-adrenergic stimulation of cyclic AMP accumulation, whereas hybridization with fat cell ghosts of hypothyroid rat does not restore this response. Ns of pigeon erythrocyte membranes radiolabeled with cholera toxin and [32P]NAD+, extracted in cholate, and reconstituted with fat cell membranes interacts with fat cell R. The ability of R to interact with Ns of pigeon erythrocyte membranes is impaired when the reconstitution is performed with membranes from the hypothyroid rat fat cell. Hypothyroidism appears to affect the ability of R to interact productively with Ns, without affecting either R number or Ns structure and function.     

Adenosine deaminase normalizes cyclic AMP responses of hypothyroid rat fat cells to forskolin, but not beta-adrenergic agonists

Lipolysis and cyclic AMP accumulation in response to beta-adrenergic agonists or forskolin are severely impaired in fat cells from the hypothyroid rat. Incubating hypothyroid rat fat cells with adenosine deaminase normalizes the cyclic AMP response to forskolin, but not to beta-adrenergic agonists. Increased sensitivity to adenosine action in the hypothyroid state appears to be the basis for the impaired cyclic AMP response to forskolin, but does not appear to be the underlying defect responsible for the impaired response to beta-adrenergic agonists.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Study of propidium iodide binding to DNA in intact cells by flow cytometry.

We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.