Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.     

Computer determination of perfusion patterns in pulmonary capillary networks

Hanger, Christopher C., Robert G. Presson, Jr., Osamu Okada,Steven J. Janke, John J. Watkins, Wiltz W. Wagner, Jr., and Ronald L. Capen. Computer determination of perfusion patterns in pulmonarycapillary networks. J. Appl. Physiol.82(4): 1283-1289, 1997.Individual pulmonary capillaries are notsteadily perfused. By using in vivo microscopy, it can readily bedemonstrated that perfusion continually switches between capillarysegments and between portions of the network within a single alveolarwall. These changes in capillary perfusion occur even when upstream pressure and flow are constant. Flow switching between capillary segments in the absence of hemodynamic changes in large upstream vessels suggests that capillary perfusion patterns could be random. Tocalculate the probability that perfusion patterns could occur bychance, it is necessary to know the total number of possible perfusionpatterns in a given capillary network. We developed a computer programthat can determine every possible perfusion pattern for any givencapillary network, and from that information we can calculate whetherperfusion of individual segments in the network is random. With theresults of the computer program, we have obtained statistical evidencethat some capillary segments in a network are nonrandomly perfused.

     

Oscillations of membrane potential in L cells

Summary Effects of divalent cations on oscillations of membrane potentials (i.e., spontaneous repetitive hyperpolarizing responses) and on hyperpolarizing responses induced by electrical stimuli as well as on resting potentials were studied in large nondividing L cells. Deprivation of Ca²⁺ from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential. Sr²⁺ or Mn²⁺ applied to the external medium in place of Ca²⁺ was able to substitute for Ca²⁺ in the generation of hyperpolarizing responses, while Mg²⁺, Ba²⁺ or La³⁺ suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca²⁺, Sr²⁺, Mn²⁺ or La³⁺ induced membrane hyperpolarization. When the external Ca²⁺, Sr²⁺ or Mn²⁺ concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca²⁺ was of the same order of magnitude as those of hyperpolarizing responses and was dependent on the external K⁺ concentration. In the light of these experimental observations, it was deduced that the K⁺ conductance increase associated with the hyperpolarizing excitation is the result of an increase in the intracellular concentration of free Ca²⁺ mainly derived from the external solution.     

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that had fused with only one erythrocyte ghost were separated in a fluorescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell.This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A-176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100–200 fold excess of fragment A-176 was needed to kill the cells.     

The disappearance rate of intraventricular bradykinin in the brain of the conscious rat

To explore the possibility that cyclic nucleotides control green algal growth and division, $\text{Chlamydomonas}$ chemostat cultures were assayed for cyclic nucleotides. Substantial qualities of cAMP were found in cells and in extracellular millieu. Most cGMP molecules were extracellular. Slowing cell growth by slowing chemostat dilution caused reversible changes in cellular morphology and cyclic nucleotide levels. During slowed growth cAMP level increased dramatically; cGMP level decreased. New cells resulting from division were not released from original cell wall.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.