A New Bacterial Agglutinin from Soybean: I. ISOLATION, PARTIAL PURIFICATION, AND CHARACTERIZATION

A new bacterial agglutinin was isolated from seeds of the soybean cultivar Clark. Purification was carried out by ammonium sulfate precipitation and ion-exchange chromatography. The agglutinin is a heat-labile glycoprotein most active at pH 4.0. Addition of Ca²⁺, Mn²⁺ and Mg²⁺ did not enhance the agglutinating activity of this glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate showed that the agglutinin is composed of two subunits of approximately 50,000 daltons each. In the undissociated state, it agglutinates Xanthomonas phaseoli var. sojensis, the causal agent of bacterial pustule disease of soybean, at concentrations as low as 10 micrograms protein per milliliter but has no hemagglutinating activity. The agglutinin could be distinguished from previously reported soybean lectins on the basis of solubility in ammonium sulfate, lack of hemagglutinating activity, molecular weight, hapten specificity, and immunological determinants.     

EVIDENCE OF A ROLE OF HETEROCYSTS IN THE SPORULATION OF A BLUE-GREEN ALGA

Sporulation of vegetative cells adjacent to heterocysts is prevented by detachment of the two cell types, probably without impairment of the capacity of those vegetative cells to sporulate. Apparently, therefore, heterocysts are in part responsible for the sporulation of vegetative cells attached to them.     

Three-dimensional Structure of Saccharomyces Invertase: ROLE OF A NON-CATALYTIC DOMAIN IN OLIGOMERIZATION AND SUBSTRATE SPECIFICITY*

Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.     

EVIDENCE FOR PREDATION AS A FACTOR IN DETERMINING SHELL COLOR FREQUENCIES IN A MANGROVE SNAIL LITTORINA SP. (PROSOBRANCHIA: LITTORINIDAE)

It was hypothesized that in Littorina populations living on Avicennia marina in Moreton Bay, Queensland, yellow shelled individuals are at a selective advantage over other shell colors and that this advantage is due to differential selection by predators. Yellow shelled individuals were more likely to be recaptured than others, indicating a higher survival rate of yellows. When predation was restricted on ten mangrove trees, the apparent advantage of yellow shells was removed. After 18 months, the relative frequency of yellow shelled individuals was significantly lower on experimental trees than on control trees. A combination of selection for crypsis and for the less common morph is suggested as the mechanism maintaining the high levels of variation in this species.     

INHIBITION OF NITRIFICATION BY CLIMAX ECOSYSTEMS. II. ADDITIONAL EVIDENCE AND POSSIBLE ROLE OF TANNINS

The same nine plots were used in this study as in our previous study on inhibition of nitrification (Rice and Pancholy, 1972). These consisted of three stands representing two stages of old field succession and the climax in each of three vegetation types in Oklahoma: tall grass prairie, post oak-blackjack oak forest, and oak-pine forest. Soil samples were analyzed three times during the growing season of 1972 for exchangeable ammonium nitrogen, nitrate, and numbers of Nitrosomonas and Nitrobacter. Results were similar to those obtained during the entire year of 1971. The amount of ammonium nitrogen was lowest in the first successional stage, intermediate in the intermediate successional stage, and highest in the climax. The amount of nitrate was highest in the first successional stage, intermediate in the intermediate successional stage, and lowest in the climax. The numbers of nitrifiers were highest in the first successional stage usually and decreased to a very low number in the climax. These data furnish additional evidence that the nitrifiers are inhibited in the climax so that ammonium nitrogen is not oxidized to nitrate as readily in the climax as in the successional stages. This would aid in the conservation of nitrogen and energy in the climax ecosystem. Some inhibition of nitrification occurred in the intermediate stage of succession also. Previous studies of tannins indicated that these are inhibitory to nitrification, so all important plant species in the intermediate successional stage and the climax were analyzed for total tannin content. A method for extracting and quantifying condensed tannins from soils was developed and the amounts of tannins were determined in each 15-cm level down to 60 cm in the same two plots in each vegetation type. Gallic and ellagic acids, which result from the digestion of hydrolyzable tannins in oak species, were also extracted and quantified in the climax oak-pine forest. All the important herbaceous species, including the grasses, were found to have considerable amounts of condensed tannins. The highest amounts of tannins occurred in the oaks and pine, however. Condensed tannins, hydrolyzable tannins, ellagic acid, gallic acid, digallic acid, and commercial tannic acid (hydrolyzable tannin), in very small concentrations, were all found to completely inhibit nitrification by Nitrosomonas in soil suspensions for 3 weeks, the duration of the tests. Slightly larger concentrations were required to inhibit nitrification by Nitrobacter under similar conditions. The concentrations of tannins, gallic acid, and ellagic acid found in the soil of the research plots were several times higher than the minimum concentrations necessary to completely inhibit nitrification. The inhibition of nitrification was always greater in the climax stand than in the intermediate successional stage in each vegetation type, and the concentration of tannins in the top 15 cm of soil was always higher in the climax stand than in the intermediate successional stage. Moreover, the amounts of tannins calculated to be added to each plot each year are much less than the amounts found in the soil, indicating that the tannins accumulate over a period of time. Thus, it appears that the tannins and tannin derivatives may play a continuous and rather prominent role in the inhibition of nitrification by vegetation.     

THE ORIGIN OF SPECIFICITY BY MEANS OF NATURAL SELECTION: EVOLVED AND NONHOST RESISTANCE IN HOST–PATHOGEN INTERACTIONS

Most species seem to be completely resistant to most pathogens and parasites. This resistance has been called “nonhost resistance” because it is exhibited by species that are considered not to be part of the normal host range of the pathogen. A conceptual model is presented suggesting that failure of infection on nonhosts may be an incidental by‐product of pathogen evolution leading to specialization on their source hosts. This model is contrasted with resistance that results from hosts evolving to resist challenge by their pathogens, either as a result of coevolution with a persistent pathogen or as the result of one‐sided evolution by the host against pathogens that are not self‐sustaining on those hosts. Distinguishing evolved from nonevolved resistance leads to contrasting predictions regarding the relationship between resistance and genetic distance. An analysis of cross‐inoculation experiments suggests that the resistance is often the product of pathogen specialization. Understanding the contrasting evolutionary origins of resistance is critical for studies on the genetics and evolution of host–pathogen interactions in human, agricultural, and natural populations. Research on human infectious disease using animal models may often study resistances that have quite contrasting evolutionary origins, and therefore very different underlying genetic mechanisms.     

Studies on Foliar Penetration: 2. THE ROLE OF LIGHT IN DETERMINING THE PENETRATION OF 2,4 DICHLOROPHENOXYACETIC, ACID

A further study has been made of the factors determining thelevel of penetration of 2,4-dichlorophenoxyacetic acid (2,4-D)into leaves. The technique involves the use of leaf disks and2,4-D containing carbon-14 in the carboxyl group. For Phaseolus vulgaris the influence of the pH of the appliedsolution is greater in the light than in the dark. Between 0and 1,000 f.c. at 27° C there is a small increase in therate of penetration into the abaxial surface. Under these conditionsthe rates remain constant up to 56 hours. This linear relationshipholds for concentrations ranging from 100 to 1,000 mg/l, andthe rate of penetration is directly proportional to the concentration.For intensities in excess of 1,000 f.c. the light response ismarkedly different: over the first few hours there is a steadyand relatively slow rate of penetration which is followed bya second phase when the rate is greatly accelerated. This acceleratedrate can be reversed by transferring the disks to darkness,and does not take place at 1° C. Likewise, if excised disksare left for more than one hour in either the light or the darkbefore applying 2,4-D then there is subsequently no phase ofaccelerated penetration. The course of penetration into theadaxial surface exhibits no accelerated rate, and compared tothe abaxial surface the rates are lower. For leaves of Ligustrum ovalifolium, which lack stomata on theadaxial surface, the rates of penetration at 27° C intoboth surfaces remain constant in either light or darkness. Forboth the adaxial and abaxial surfaces, the rate progressivelyincreases from 0 to; 2000 f.c. and there is no phase of acceleratedpenetration. Penetration into the adaxial surface is less. At1° C the rates for both surfaces in either light or darknessare depressed. If disks of Phaseolus are irradiated with ultraviolet light,subsequent penetration is markedly depressed in the light at27° C, but in the dark or at 1° C it is enhanced. On the basis of these findings it is concluded that both physicaland metabolic factors control the rate of penetration of 2,4-DTransport through the cuticle will be dependent on adsorptionand the length of the diffusion paths. Once diffusion gradientshave been established between the surface of the cuticle andthe outer surface of the cytoplasm in the epidermal cells, thesteepness will be dependent on the rates at which 2,4-D is eitherconverted into some metabolite or moved away from the surfaceor into other cells. The relative importance of physical andmetabolic process will be dependent on the permeability andthickness of the cuticle and the level of metabolic activity.The possible role of ectodesmata in determining both the lengthand steepness of the diffusion paths is discussed.     

EVOLUTIONARY MODIFICATION IN CLARKIA. II. ROLE OF FLOWER BUD PUBESCENCE IN SECTION PHAEOSTOMA

Observations of the habitats and relative flowering of a Clarkia species with hairy flower buds and several with hairless flower buds led to the hypothesis that long hairs on flower buds regulate bud temperature. This hypothesis predicts that hairless buds would be warmer and develop faster than hairy buds, which would be cooler, develop more slowly, and avoid high temperature stress. The hypothesis was tested by comparing flower bud growth rates and temperatures in three genetically similar biotypes of Clarkia unguiculata and in all six species of section Phaeostoma. Flower buds of the three biotypes included hairy (HY) and hairless (HN) from the same coastal population and densely hairy (HD) from an interior locality. The six species included C. unguiculata with densely hairy buds (HD) and five related species with hairless buds. Contrary to expectations, HY buds grew more rapidly than HN buds. HD buds grew more rapidly than either and also more rapidly than the hairless buds of five related species. Again contrary to expectations, the three biotypes of C. unguiculata had equivalent temperature relations, with bud temperatures mostly somewhat below air temperatures. In a comparative experiment, bud temperatures in C. unguiculata approximated air temperatures while bud temperatures in five related species mostly fell well below air temperatures. Thus, predictions of the hypothesis were not borne out. Long bud hairs apparently have minimal effect on bud growth rates and temperatures, and we conclude that physiological adaptations are more important. Bud cooling mechanisms are discussed.     

SEXUAL SELECTION AGAINST HYBRIDS BETWEEN SYMPATRIC STICKLEBACK SPECIES: EVIDENCE FROM A FIELD EXPERIMENT

Sexual selection against viable, fertile hybrids may contribute to reproductive isolation between recently diverged species. If so, then sexual selection may be implicated in the speciation process. Laboratory measures of the mating success of hybrids may underestimate the amount of sexual selection against them if selection pressures are habitat specific. Male F₁ hybrids between sympatric benthic and limnetic sticklebacks (Gasterosteus aculeatus complex) do not suffer a mating disadvantage when tested in the laboratory. However, in the wild males choose different microhabitats and parental females tend to be found in the same habitats as conspecific males. This sets up the opportunity for sexual selection against male hybrids because they must compete with parental males for access to parental females. To test for sexual selection against adult F₁ hybrid males, we examined their mating success in enclosures in their preferred habitat (open, unvegetated substrate) where limnetic males and females also predominate. We found significantly reduced mating success in F₁ hybrid males compared with limnetic males. Thus, sexual selection, like other mechanisms of postzygotic isolation between young sister species, may be stronger in a wild setting than in the laboratory because of habitat-specific selection pressures. Our results are consistent with, but do not confirm, a role for sexual selection in stickleback speciation.     

ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles: EVIDENCE FOR HEPARAN SULFATE EPITOPE SPECIFICITY AND ROLE OF BOTH SYNDECAN AND GLYPICAN*

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (αHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [³⁵S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.     

Synthesis of Nitrate Reductase in Chlorella: II. EVIDENCE FOR SYNTHESIS IN AMMONIA-GROWN CELLS

Antiserum was prepared against nitrate reductase (EC 1.6.6.1) purified to homogeneity from Chlorella vulgaris Beijerinck. Both crude antiserum and anti-nitrate reductase antibodies prepared from it were used as re-agents to study the synthesis of nitrate reductase. Cell extracts from cultures which were grown with ammonia salts as the sole source of nitrogen contained almost no active enzyme. These extracts did contain material which binds to antibody and is thus immunologically related to purified nitrate reductase. The presence of this cross reacting material in cell extracts was detected by the ability of these extracts to (a) lower the titer of antisera; (b) form a biphasic precipitin curve with purified antibody; and (c) increase the peak height of a standard amount of purified nitrate reductase in rocket immunoelectrophoresis assay. These results suggest that ammonia-grown cells contain nitrate reductase precursor protein.     

Hormonal Regulation of Hypocotyl Elongation in Lactuca sativa L.: I. EVIDENCE AGAINST THE INVOLVEMENT OF GIBBERELLINS IN THE ELONGATION OF HYPOCOTYL IN SEEDLINGS GROWN IN THE DARK

The kinetics of extension induced by GA₃¹ in the hypocotyl ofintact seedlings of Lactuca sativa are similar in the dark andin the light, and differs fundamentally from the kinetics ofelongation in the dark without GA₃. Both in continuous lightand in the dark, GA₃-induced promotion starts 24 h after incubation.In the dark, even low concentrations of GA₃, which do not affectthe length measured after 6 d when the extension of hypocotylalmost ceases, remove the lag period of 48 h which precedesextension, and prolong the high rate of elongation. FollowingGA₃ supply the hypocotyl length in the dark and in the lightdoes not differ until 48 h; thereafter the rate of elongationin the light is less, so that the final length of the hypocotylis 40 per cent shorter than that of the dark-grown seedlingswithout GA₃. IAA supplied apically to light-grown seedlings induces a weakpromotion at a concentration of 1 mg l^–1 only. With anincreasing concentration of GA₃ supplied simultaneously, theconcentration of IAA inducing a significant promotion decreases.A combined supply of both these regulators, however, does notrestore the light-mediated inhibition of hypocotyl elongationcompletely. The maximum decrease in hypocotyl length induced by the growthretardants AMO-1618, CCC, and B-9 supplied from the beginningin the dark does not exceed 70 per cent. Saturating doses ofGA₃ supplied in combination with any one of the retardants compensateonly a fraction of the decrease. The results have been interpreted to show that native GAs arenot involved in extension growth in the dark.