Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex.

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.     

Influence of oral BM 12531 (INN azimexon) on immune response in untreated cancer patients

Summary To evaluate the immune-stimulating properties of 2-(2-cyanaziridinyl-[1]-)-2-(2-carbamoylaziridinyl-[1]-) propane (INN azimexon) a randomized double-blind study was performed in 30 cancer patients with different solid tumors. A group of 15 patients received 300 mg azimexon PO over a 7-day period, while 15 other patients received a placebo. We could not demonstrate an increase of total or active T lymphocytes, and the ³H-thymidine uptake of isolated lymphocytes with or without PHA stimulation was not increased when tested 3, 7, 14, and 21 days after the beginning of therapy. The serum concentration of IgM showed a slight but not significant increase, while IgG, IgA, C₃, and C₄ levels remained unchanged. The major side-effect was a significant decrease of hemoglobin and red blood cell count after azimexon administration, which was even more pronounced in four patients given 900 mg azimexon.     

ROLE OF MYCORRHIZAL FUNGUS ORIGIN IN GROWTH AND NUTRIENT UPTAKE BY GERANIUM ROBERTIANUM

Prior field studies have shown that populations of forest herbs on relatively nutrient poor soils have higher vesicular-arbuscular mycorrhizal (VAM) infection intensity than plants on rich soils. However, the growth responses and ability to take up P against the soil nutrient gradient are often not linearly related to infection intensity. To determine if intraspecific differences among populations of the common VAM fungus Glomus occultum could differentially affect growth and nutrient uptake, Geranium robertianum seedlings were inoculated with Glomus occultum isolated from four forest types along a gradient of soil fertility, and grown in a greenhouse at P levels typical of the extremes of that gradient. Plants given inoculum from relatively infertile forest sites generally produced greater root, shoot, and total mass than plants given inoculum from fertile sites or uninoculated plants, especially at the low P supply rate. Total P uptake and both P and N uptake efficiency were also highest in plants given inocula from low fertility sites. These results indicate that local adaptation and intraspecific variations in the ability of VAM fungi to induce growth and nutrient uptake effects on host plants may be as important as interspecific differences among VAM fungus species.     

Enzymatic synthesis and inhibitory characteristics of tartronate semialdehyde phosphate

The immediate product of the pyruvate kinase catalyzed phosphorylation of beta-hydroxypyruvate is the enol of tartronate semialdehyde phosphate (TSP). The reaction has the same pH profile as that for the phosphorylation of pyruvate with pK's of 8.2 and 9.7 observed in H2O. This enol tautomerizes in solution to the aldehyde, which in turn becomes hydrated. 31P NMR spectra indicate that the enol resonates approximately 1 ppm upfield from the hydrated aldehyde. By following the tautomerization spectrophotometrically at 240 nm, we have found it to be independent of pH (0.2 min-1 below pH 6 in water), except that it is 2-fold slower above the pK of the phosphate group (6.3 in H2O and 6.7 in D2O). It is 3.6-fold slower in D2O. When this TSP is reduced with NaBH4, approximately 50% of the product is D-2-phosphoglyceric acid (substrate for enolase). Thus, while the immediate product of the phosphorylation rection is the enol of TSP, the eventual product is D,L-TSP. Both the enol and the aldehyde forms of TSP were found to be potent inhibitors of yeast enolase with apparent Ki values of 100 nM and 5 microM, respectively. However, since the aldehyde form is 95-99% hydrated [Stubbe, J., & Abeles, R. (1980) Biochemistry 19, 5505], the true Ki for the aldehyde species is 50-250 nM. The enol of TSP shows slow binding behavior, as expected for an intermediate analogue, with a t1/2 for this process of approximately 15 s (k = 0.046 s-1) and an initial Ki of approximately 200 nM.     

Vegetation boundary detection: A comparison of two approaches applied to field data

We examined and compared two approaches to vegetation boundarydetermination by applying them to field data collected from Betsch Fen, analkaline wetland in Ohio, USA. Two boundary detection methods were used to testhypothesized boundary locations determined through field observations: gradientanalysis by detrended correspondence analysis (DCA) and the moving split-window(MSW) technique. These two methods represent approaches suggested by vegetationanalysis and landscape ecological literature, respectively. DCA was moresuccessful in detecting vegetation changes at the community level, but it wasoften difficult to extrapolate this information to a landscape context. Incontrast, MSW detected changes at a landscape level which overestimated minorshifts in species composition at the community level. Although results from MSWwere more easily interpretable, neither method completely confirmedhypothesizedboundary locations derived from field reconnaissance and aerial photo analysis.While DCA and MSW can be used in conjunction to provide maximum information onboundary location and ecological significance, this would be unrealistic inpractical applications because of the time and effort required to do bothsimultaneously. We suggest an approach using primarily MSW principles, whichtake into consideration community-level information and optimum sampling plotplacement and spacing.     

Effects of Ca supply and stress on uptake and translocation of Ca in two poinsettia cultivars

Distortion of bracts and leaves of poinsettia (Euphorbia pulcherimma Willd.) is considered to be a symptom of Ca deficiency. This study was designed to determine whether rates of either Ca uptake or translocation, or responses to interactions between Ca uptake/ translocation and transpirational stress differed between the distortion-susecptible “V-14 Glory” cultivar and the non-susceptible “Annette Hegg Dark Red (AHDR)” cultivar. Ca uptake increased linearly with Ca supply in the aerated nutrient solution in both cultivars, but did not differ between cultivars. Strong transpirational stress applied for 24 hr did not significantly reduce Ca uptake measured 1 and 14 days later in either cultivar. Roots had significantly lower Ca tissue concentrations than shoots in both cultivars, and Ca tissue concentrations tended to be higher in the mid-portion of the shoot than in the basal or apical sections. As no clear differences in Ca uptake or translocation between susceptible and non-susceptible cultivars were observed, we suggest that the Ca deficiencies which produce bract/leaf distortion must be localized and produced by within-leaf or -bract processes. Furthermore, transient though intense transpirational stresses are insufficient to affect subsequent Ca uptake in these cultivars.     

Mycorrhizal dynamics of three woodland herbs of contrasting phenology along topographic gradients

Vesicular-arbuscular mycorrhizal (VAM) dynamics of three woodland herbs of differing phenology were examined along contiguous topographic gradients in two southwestern Ohio forests during 1992-1994. Cardamine concatenata (Brassicaceae) exhibited VAM colonization only during senescence in May, whereas Trillium flexipes and Smilacina racemosa exhibited robust VAM development throughout the year. The lowest mean percent root length colonized (PRLC) for T. flexipes and S. racemosa was 38.3% and 37.7%, respectively, while the greatest was 80.2% and 80.8%, respectively. Overall, analysis of variance (ANOVA) indicated significant seasonal effects with summer months having higher mean PRLC, and winter and early spring months having the lowest. ANOVA also indicated a significant effect due to topography, with uplands and slopes having greater mean PRLC than lowlands. The significantly lower mean PRLC for both T. flexipes and S. racemosa may have reflected higher phosphate availability in the spring, cooler temperatures, and/or moister conditions. The significantly lower mean PRLC in lowland positions may be due to greater soil moisture throughout the year. These results support the hypothesis that VAM development is lower in moister topographic positions, however, they do not support the hypothesis that VAM development would increase with decreasing P availability. Consequently, moisture appeared more important than P in regulating VAM development in these sites.