Single potassium channels in membranes of isolated mesophyll barley vacuoles

Summary Voltage-dependent K channels could be identified in on-cell and excised patch-clamp records on membranes of isolated plant cell vacuoles. The current through a membrane patch is dominated by a channel population with a conductance of about 121 pS in symmetrical 250mm KCl solution. The single channel adopts at least two conducting levels the 121-pS state being most frequently observed. The channel shows outward rectification, representing a cation flux into the vacuoles. The rectification appears to be caused by a vanishing open probability and a short channel lifetime at hyperpolarizing voltages. A selectivity ratio of potassium over sodium of about 6 was derived as an estimate. Occasionally, an additional population of K channels with a single-channel conductance of approximately 18 pS is observed. This channel type exhibits outward rectification as well.     

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

Prediction of F3 row performance from F2 individual plant data in oats

Summary Parameters estimated from a Gardner-Eberhart analysis of the F₂ generation of a six-parent diallel in oats (Avena sativa L.) were used to compare methods for predicting the performance of F₃ row plots. The prediction methods were: (1) individual F₂ plant performance (F2I), (2) parent average plus F₂ plot deviations (PF2), (3) parent average plus weighted F₂ plot deviations (PF2P), (4) best linear unbiased prediction (BLUP) of parent average plus F₂ plot deviations (BPF2), and (5) BLUP plus weighted F₂ deviations (BF2). The F₂ single-plant traits used for prediction were biological yield to predict F₃ biological yield, whole plant and primary tiller grain yield for prediction of F₃ grain yield, and whole plant and primary tiller harvest index (HI) to predict F₃ HI. Prediction methods were evaluated by correlations between predicted and observed F₃ performance. Prediction methods and traits for which correlations were greater than for F2I included: BF2 for biological yield, PF2, PF2P and BF2 for whole plant grain yield, PF2, BPF2, and BF2 for primary tiller grain yield. None had a correlation significantly greater than F2I for either measure of HI, where heritability was large. PF2 is the recommended method for traits with low heritability because of its simplicity and because it had the largest or nearly the largest correlation for each of the yield traits. F2I is the recommended method for traits with larger heritability.Contribution No. 8821 of the U.S. Regional Pasture Research LaboratoryDeceased     

Metabolism of semisynthetic single-chain GM1 derivatives in cerebellar granule cells in culture

Semisynthetic single-chain GM1 derivatives containing N-acetyl-sphingosine (LIGA4) or N-dichloroacetyl-sphingosine (LIGA20) were recently reported to exert strong protection against glutamate-induced neuronal death in primary cultures of cerebellar granule cells. Elucidation of the molecular mechanism underlying the evoked effect requires knowledge of the metabolic fate of such molecules in the same cultured cells. For this, LIGA4 and LIGA20 were made radioactive on the long chain base moiety and added to cerebellar granule cells in culture in parallel with GM1 ganglioside. The metabolic fate was then investigated. It was found that both these molecules were easily taken up by the cells and promptly metabolized in a fashion qualitatively similar to that of control GM1. The highest amount processed was attributed to the different aggregation properties of LIGAs in solution. Among metabolites, higher accumulation of the single-chain ceramide residues was found after LIGA administration. Interestingly, sphingomyelin was generated, regardless the added compound, suggesting a recycling of the free long chain base.     

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).     

Induction of tumor cell resistance to macrophage-mediated lysis by preexposure to non-activated macrophages

Thioglycollate-elicited peritoneal exudate (non-activated) macrophages do not lyse tumor cells and in contrast to activated macrophages bind less target cells. However, a non-lethal encounter of tumor cells with non-activated macrophages resulted in a pronounced effect on the subsequent tumor cell binding to and lysis by activated macrophages. Our results have shown that binding of tumor cells by non-activated macrophages was Ca2+ and temperature dependent; had a requirement for a Pronase-sensitive structure on macrophage surface membranes; was saturable; and was 2-3X less than that observed for activated macrophages. Experiments were conducted in which syngeneic tumor cells were incubated with a monolayer of non-activated macrophages and then assayed for selective binding and sensitivity to lysis. The important observations were that as a result of a 3-hr incubation with non-activated macrophages at an EC: TC ratio of 5:1 there was an increase in the number of tumor cells that bound to both activated and non-activated macrophages; a loss of selective binding in which the ratio of tumor cells bound to activated/non-activated macrophages (normally greater than 2) was lowered to 1.0; and a concomitant decrease in the susceptibility of tumor cells to macrophage-mediated cytolysis. The induction of tumor cell resistance to macrophage kill required an exposure to an excess number of non-activated macrophages, was reversible upon culturing with or without macrophages for 24 hr and required cell-cell contact. Our results reinforce the importance of selective binding between tumor cells and activated macrophages as an initial phase in tumor cell killing and also illustrates an active role for non-activated macrophages in vivo in allowing tumor cells to escape the immune surveillance by activated macrophages.     

Characterization of a Ca-dependent maxi K channel in the apical membrane of a cultured renal epithelium (JTC-12.P3)

Summary A Ca and potential-dependent K channel of large unit conductance was detected in the apical membrane of JTC-12.P3 cells, a continuous epithelial cell line of renal origin. The open probability of the channel is dependent on membrane potential and cytoplasmic-free Ca concentration. At cell-free configuration of the membrane patch, the open probability shows a bell-shaped behavior as function of membrane potential, which decreases at larger depolarization. With increasing Ca concentration, the width of the bell-shaped curve increases and the maximum shifts into the hyperpolarizing direction. For the first time the kinetics of this channel was analyzed under cell-attached conditions. In this case the kinetics could sufficiently be described by a simple open-closed behavior. The channel has an extremely small open probability at resting potential, which increases exponentially with depolarization. The low probability induces an uncertainty about the actual number of channels in the membrane patch. The number of channels is estimated by kinetic analysis. It is discussed that this K channel is essential for the repolarization of the membrane potential during electrogenic sodium-solute cotransport across the apical membrane.     

Lack of evidence for liver lectins functioning as IgM or IgG-Fc-receptors

We have tested whether mannose- and galactose-specific lectins on liver cells are able to bind antibody-antigen complexes and thus function as Fc-receptors. Rat hepatocytes and liver sinusoidal cells were isolated by collagenase perfusion and differential centrifugation. Rat erythrocytes were coated with purified IgM or IgG from rabbits immunized with rat erythrocytes. Both IgM and IgG coated erythrocytes bound to liver macrophages but not to hepatocytes. The binding of IgM and IgG coated red blood cells to liver macrophages could not be blocked by potent inhibitors for mannose- and galactose-specific macrophage lectins such as mannan, D-mannose-bovine serum albumin, N-acetyl-D-galactosamine, D-galactose-bovine serum albumin, or asialofetuin. Although lectin activity is calcium dependent and trypsin sensitive neither condition blocked rosette formation between liver macrophages and opsonized erythrocytes. Thus mannose- and galactose-specific lectins are not involved in the sequestration of IgM- or IgG-antibody-erythrocyte complexes in the liver.     

Morphological transformation, autonomous proliferation and colony formation by chicken heart mesenchymal cells infected with avian sarcoma, erythroblastosis and myelocytomatosis viruses

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses probably reflects differences in the modes of action of the transforming proteins encoded by the onc genes of the respective viruses.(ABSTRACT TRUNCATED AT 400 WORDS)