Muscarinic stimulation of inositol phosphate accumulation and acid secretion in gastric fundic mucosal cells

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time- and dose-dependent: EC50 values for IP1, IP2 and IP3 accumulation were not statistically different. The mean value was 30 +/- 8 microM (n = 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 microM CCh was 160 +/- 15%. CCh-induced IP accumulation was abolished by atropine (Ki = 0.32 +/- 0.18 nM (n = 3)). The CCh concentrations leading to half-maximal inhibition of N-[3H]methylscopolamine binding and half-maximal IP accumulation were similar. The half-maximal value for CCh-induced aminopyrine accumulation was 8-times lower. These results indicate that IP3-mediated mobilization of intracellular Ca2+ might be involved in CCh-induced acid secretion by parietal cells.     

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.     

Mitogenic effect of PMA + IL 2 on subpopulations of corticoresistant thymocytes

The proliferative response of subpopulations of corticoresistant thymocytes (CRT) to phorbol-12-myristate-13-acetate (PMA) + interleukin 2 (IL 2) was investigated. Thymocyte subpopulations were selected by the indirect "panning" technique, and their purity was checked by cytofluorometry. Microcultures were set up with an optimal concentration of PMA, EL4 supernatant, or pure IL 2 obtained by recombinant DNA technology (r-IL 2) in the presence or in the absence of accessory splenic adherent cells (SAC). Under these conditions, only the Lyt-2+ CRT proliferated, and this response was IL 2-dose-dependent and was increased by accessory cells. When the calcium ionophore A23187 was added to the cultures, the proliferation of L3T4+ CRT was greatly increased. These results were confirmed by cultures at limiting dilution of positively selected Lyt-2+ and L3T4+ subpopulations of CRT at optimal concentrations of PMA, r-IL 2, A23187, and accessory cells. These results are consistent with the idea that two signals are necessary to activate L3T4+ CRT, whereas only IL 2 is necessary for PMA-induced proliferation of Lyt-2+ CRT. Finally, unlike the case of lectin-induced proliferation of Lyt-2+ and L3T4+ CRT, the presence of accessory cells or cell-cell contact is important for optimal response to PMA + IL 2.     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.     

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.     

Regulation of Gene Expression in Developing Zea mays Embryos: Protein Synthesis during Embryogenesis and Early Germination of Maize

Polypeptides synthesized in dissected embryos of Zea mays at different stages of embryogenesis and early germination have been characterized by their migration in two-dimensional gel electrophoresis. This analysis has been carried out with in vivo labeled polypeptides from excised embryos and with proteins synthesized in vitro in the rabbit reticulocyte system directed by poly(A⁺) RNA isolated from the different developmental stages. We have identified three main sets of expressed polypeptides: (a) embryonic set: this group of polypeptides is synthesized in young and mature embryos but not in early germination; (b) maturation set: this group of polypeptides is not present in young embryos and appears during the maturation period. Some of these polypeptides are still present in early germination while others disappear from stored mRNAs in dry embryos. One particular group from this set can be induced prematurely in young embryos by incubation with abscisic acid; and (c) germination set: this group of polypeptides is not expressed in the maturation period and appears after brief imbibition of the dry embryos.     

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self-reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes.     

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.     

Specific recognition by the tripeptide lysyl-tryptophyl-alpha-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558-563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by trans Pt are required to observe a change in stacking efficiency. In contrast modification by cis Pt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cis Pt.