Specific interaction of 5-(N-methyl-N-isobutyl)amiloride with the organic cation-proton antiporter in human placental brush-border membrane vesicles. Transport and binding.

The interaction of 5-(N-methyl-N-isobutyl)amiloride (MIBA) with brush-border membrane vesicles isolated from normal human term placentas was investigated using two parameters: binding and transport. The binding of MIBA to placental membranes was specific and temperature- and pH-dependent, and the apparent dissociation constant (Kd) for the process was 58 +/- 2 microM. The binding was inhibited by other amiloride analogs and also by clonidine and cimetidine with a rank order potency: MIBA > benzamil > dimethylamiloride > amiloride > clonidine > cimetidine. These compounds also inhibited Na(+)-H+ exchanger activity in these membrane vesicles, but with a different order of potency: dimethylamiloride > MIBA > amiloride > benzamil > cimetidine > clonidine. The membrane vesicles were also able to transport MIBA into the intravesicular space, and the transport was stimulated many-fold by the presence of an outwardly directed H+ gradient across the membrane. The H+ gradient was the driving force for uphill accumulation of MIBA inside the vesicles. The transport process was electrically silent. The transport of MIBA was inhibited by other amiloride analogs and by clonidine and cimetidine, and the order of potency was the same as the order with which these compounds inhibited the binding of MIBA. The Michaelis-Menten constant (Kt) for the transport process was 46 +/- 2 microM. The binding as well as the transport were also inhibited by Na+ and Li+. Interestingly, tetraethylammonium and N1-methylnicotinamide, two of the commonly used substrates in organic cation transport studies, failed to inhibit the binding and transport of MIBA. Furthermore, although the outwardly directed H+ gradient-dependent uphill transport of tetraethylammonium could be demonstrated in renal brush-border membrane vesicles, there was no evidence for the presence of a transport system for this prototypical organic cation in placental brush-border membrane vesicles. It is concluded that the human placental brush-border membranes possess an organic cation-proton antiporter which accepts MIBA as a substrate, the low affinity binding site for MIBA observed in these membranes represents this antiporter, and that the placental organic cation-proton antiporter is distinct from the widely studied renal organic cation-proton antiporter.     

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison.

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150-200 and 1421-1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.     

Rapid Identification of Viable Bacterial Spores Using a Fluorescence Method

A method has been devised to differentiate viable and nonviable bacterial spores. “Germination-like” changes are initiated in spores with performic acid and lysozyme. The germinated spores are stained with aqueous acridine orange, a fluorescent dye. Nonviable spores fluoresce lemon-green and viable spores orange-red. It is proposed that with the use of a membrane filter resistant to performic acid and lysozyme, the method may be used for spore enumeration in foods in about 4 hr compared to conventional plating methods, which usually require up to 72 hr.     

Mutagenic activity of antibiotics alone and in conjunction with alkanesulfonates

Differential and combined effects of 0.25 and 0.50% antibiotics (ampicillin, neomycin, furadentine) and alkylating agents (ethyl methanesulfonate, methyl ethanesulfonate, methyl methanesulfonate) were assayed on Phaseolus vulgaris L. (2 n = 22) at the M₂ generation for chlorophyll mutations. The general types scored were Albino, Xantha, Virescens and Maculata. Yellowish-green leaves having red mid-veins and veinlets were observed only amongst the progeny raised after treatment with 0.25% ethyl methanesulfonate or 0.25% methyl ethanesulfonate + 0.25% ampicillin. The frequency of chlorophyll mutation after combined treatments in general was higher than after differential treatments. Methyl methanesulfonate among alkanesulfonates and neomycin among antibiotics induced higher frequencies of chlorophyll mutations. No chlorophyll mutant was produced by ampicillin.Although antibiotics induced a lower frequency of chlorophyll mutation than alkylating agents, the frequency and pattern of spectra of chlorophyll mutants showed an action of antibiotics in inducing mutation similar to that of alkylating agents. Therefore, it is considered that antibiotics are potential mutagens.     

Cyclo (His-Pro): a selective inhibitor of rat prolactin secretion in vitro

Cyclo (His-Pro) (10 ng/ml), inhibits KCl (59 mM) or thyrotropin-releasing hormone (10 ng/ml) stimulated, but not basal, release of prolactin from rat hemipituitaries $\text{in vitro}$. However, cyclo (His-Pro) has no effect on the basal or stimulated release of thyrotropin and growth hormone. Cyclo (His-Pro) does not inhibit the binding of thyrotropin-releasing hormone to pituitary membrane suggesting that cyclo (His-Pro) inhibition of prolactin release is not mediated via the pituitary TRH-receptor.     

Contact inhibition of locomotion and the structure of homotypic and heterotypic intercellular contacts in embryonic epithelial cultures

Epithelia from the early chick embryo have been grown in culture and then fixed for electron microscopy so that the ultrastructure of intercellular contacts could be examined. Epithelia were used which showed various forms of contact inhibition of locomotion upon confrontation with one another. Confrontations of hypoblast with hypoblast after 6 days, and endoblast with endoblast after 24 h showed type 1 contact inhibition and formed desmosomes and zonulae adhaerentes with extensive microfilament collinearity between apposed cells. Hypoblast-hypoblast confrontations after 24 h resulted in type 2 contact inhibition with considerable ruffling and position shifting. In this case desmosomes were absent and microfilament collinearity was restricted. Endoblast cells after 24 h in culture show type 2 inhibition with respect to hypoblast monolayers which they infiltrated upon confrontation. Examination of these heterotypic contacts also showed an absence of desmosomes and reduced adhaerens junctions. Intermediate filaments accumulated at all contact sites examined. It is concluded that whereas type 1 contact inhibition of locomotion in these epithelial cells is accompanied by desmosome formation and extensive zonula adhaerens junctions, type 2 inhibition is not. These junctional deficiences may be responsible in part for the cell motility characteristically observed in monolayers of type 2 inhibited cells.     

Eocyzicus spinifer sp. nov. (Conchostraca: Cyzicidae) from Iraq

A new Conchostracan species, Eocyzicus spinifer from Iraq is described. A table is given to distinguish the known species of the genus Eocyzicus.     

Membranes in the miotic apparatus of barley cells

Membranes in the mitotic apparatus have been investigated ultrastructually in dividing cells of barley (Hordeum vulgare). After osmium tetroxide- potassium ferricyanide or ferrocyanide postfixation (OsFeCN) of material that had been fixed in glutaraldehyde in the presence of Ca(++), the nuclear envolope (NE)-endoplasmic reticulum (ER) complex is selectively stained, permitting observations on the cellular pattern and structural ramifications of this membrane system that have not been previously recognized. Specifically, it is observed that membrane system that have not been previously recognized. Specifically, it is observed that during mitosis the NE-ER forms a continuous membrane system that ensheathes and isolates the mitotic apparatus (MA). Elements of ER progressively accumulate in the region of the spindle pole, becoming most concentrated by early anaphase. Within the MA itself, there are striking spindle- membrane associations; in particular, tubular elements of predominantly smooth NE-ER invade the spindle interior selectively along kinetochore microtubules. The membrane elements at the pole and surrounding the MA consist of tubular reticulum and fenestrated lamellae. Membranes of the MA thus resemble in considerable detail the tubular network and fenestrated elements of the sarcoplasmic reticulum of muscle. It is suggested that the NE-ER of the dividing barley cell may function in one or both of the following ways: (a) to control the concentration of free Ca(++) in the MA and (b) to serve as an anchor to chromosome motion.