OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium

Summary The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Gene regulation during dedifferentiation in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire the capacity to rapidly recapitulate morphogenesis. Therefore, when cells at the loose aggregate stage are disaggregated and challenged to reaggregate, they do so in a tenth of the original time. If loose aggregate cells are disaggregated and resuspended in buffered dextrose solution (erasure medium), they retain the capacity of rapid recapitulation for 80 min, then completely lose this capacity in a single, synchronous step referred to as the "erasure event." The erasure event sets in motion a program of dedifferentiation during which cells lose developmentally acquired characteristics at different times. The erasure event is inhibited by the addition of 10(-4) M cAMP to erasure medium. The synthesis of 33 growth-associated polypeptides, the synthesis of 53 development-associated polypeptides, and the level of 2 development-associated RNAs have been monitored during the erasure program and in cultures inhibited from erasing by the addition of 10(-4) M cAMP. Growth-associated polypeptides begin to be resynthesized and development-associated polypeptides exhibit dramatic decreases in rate of synthesis at different times throughout the first 240 min in erasure medium. Inhibiting the erasure event with cAMP has no major effect in the resynthesis of the majority of growth-associated polypeptides. Only one growth-associated polypeptide, V28, is completely inhibited by cAMP, suggesting that it may play a role in the erasure process. In contrast, inhibiting the erasure event with cAMP has a marked effect on the synthesis of development-associated polypeptides, causing a dramatic reduction in the rate at which synthesis decreases for 6 polypeptides, and completely inhibits the decrease in the synthetic rate of 8 polypeptides. The two development-associated RNAs, 16G1 and 10C3, exhibit two distinctly different patterns of loss during erasure, but in both cases cAMP added at time zero of the erasure process dramatically retards or inhibits loss. In addition, when cAMP is added just prior to the erasure event, it inhibits the erasure event and stimulates a rapid increase in the level of 16G1 RNA back to the developmental level. The level of 16G1 RNA then remains at this level for at least 400 min. When cAMP is added after the erasure event, it causes a low, transient increase in the level of 16G1 RNA. These results are considered both in relation to the program of erasure, and in relation to the role of cAMP in the expression of developmental genes during the forward program of development.     

Workshop on algal polysaccharides

Summary This workshop was an impromptu event, but the fact that a number of interesting problems were identified by the participants from the floor may indicate that it is worth repeating. If it is to be repeated, however, it is important that notice be given, and that the scope of the workshop be defined in advance, so that participants can be better informed and bring supporting data. The best approach would be to identify a convenor and define the scope of the workshop prior to the first circular for the next Seaweed Symposium; invitations could then be issued by the convenor for specific topics and data, and a very brief program could be issued with the third circular. Such a procedure may very well allow the identification of new areas for research.     

Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted 113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. 113Cd resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of the resonances to the transition metal (S1) and calcium (S2) sites were unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and S2 sites, respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH fail to show a 113Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.     

A light and electron microscopic study of the quadriflagellate green algaSpermatozopsis exsultans

The green flagellateSpermatozopsis exsultans Korshikov has been studied in culture by light and electron microscopy. The organism is naked, bears four flagella and is conspicuously spirally twisted. The ultrastructure and location of cell organelles (except the flagellar apparatus) has been investigated in detail using an absolute configuration analysis. With the exception of a doubling of the flagella and of the secondary cytoskeletal microtubule system,S. exsultans has the exact same complement of organelles occupying the same relative positions as has been described forS. similis. The two species are therefore correctly placed in the same genus. The usefulness of absolute orientations of cell organelles for green algal taxonomy and phylogeny is stressed.Dedicated to Prof.M. Mix on the occasion of her 60th birthday.     

The P-M Hybrid Dysgenesis Cline in Eastern Australian Drosophila melanogaster: Discrete P, Q and M Regions Are Nearly Contiguous

The dramatic latitudinal cline in P-M hybrid dysgenesis characteristics along the east coast of Australia is not smooth. Tests of recent collections of Drosophila melanogaster from the southeastern coast define the previously described cline as comprising three discrete, apparently contiguous regions of P, Q and M phenotypes, respectively. Northern populations from Cairns (16.9°SLat) to Ourimbah (33.4°SLat) are phenotypically P; populations from Wollongong (34.4°SLat) to Eden (37.1°SLat) are Q; and populations from Genoa (37.5°SLat) to Cygnet (43.2°SLat) are M. The decline in P activity from northern Queensland (55-60% gonadal dysgenesis (GD) in cross A) to mid-New South Wales (20-30% GD in cross A) is gradual; proceeding south, there then is a sharp drop to Q populations (<10% GD in crosses A and A*). This drop in P activity occurs in only 150 km, across the urban and suburban area of Sydney. Q populations are then found south to Eden, but Genoa, only about 50 km further southeast, is clearly M (48% GD in cross A*), as are two populations further south. The two discontinuities in the P-M cline do not correspond to obvious climatic differences along the coast, nor to obvious barriers to dispersal of D. melanogaster. The cline has apparently not moved between 1983 and 1985-1986.     

Connective tissue influences on the expression of epithelial cell-surface antigens

Summary Adult mice were found to show regional variation in the epithelial expression of some molecules of the blood-group antigen series. To investigate connective tissue influences on such differences, heterotypic recombinants of epithelia and connective tissues from various regions were prepared and examined using monoclonal antibodies directed against bloodgroup antigens H and Le^y. The results indicate that epithelia may maintain a preexisting regionally specific pattern following recombination but that, in some recombinant matches, the connective tissue is capable of signalling redirection of the pattern of expression towards that typical of the epithelium with which it is normally associated.This work was supported by NIH-NIDR RO1-DEO-5190