Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

Iron kinetics effects of 88 millirads. Partial-versus-total body X-irradiation

Rats were irradiated with one tibia shielded (95% marrow exposure), total body exposed (TBI, 100%), and only one tibia exposed (5%), or they were sham irradiated (SI, 0%). Plasma Fe-59 clearance time (T1/2) and Fe-59 content ratio in the right and left tibia (RT/LT) were assayed to determine the erythroid activity of the overall marrow of the animals and the relative marrow activity in the exposed and shielded tibias, respectively. When a major fraction of the overall marrow was shielded or irradiated, the overall erythroid activity levels were identical to those of the SI and TBI animals, respectively. Interestingly, enhanced normoblastosis was observed in the marrow of the exposed tibia of individual animals exhibiting normal erythroid activity in 95% of the marrow. Conversely, localized marrow with normal erythroid activity was found in a shielded tibia of individual rats, demonstrating an enhanced erythroid activity in a major fraction of the total body. It was concluded that 88 mrad can alter marrow functions in a small isolated skeletal region as effectively as in the whole body, and tandem assays of the Fe-59 T1/2 and Fe-59 RT/LT can facilitate ultra-low-dose X-ray studies involved with partial body exposures.     

Generalizing from experiments: is predation strong or weak in the New England rocky intertidal?

Summary Petraitis (1990) recently critized previous generalizations regarding the effects of predation in the New England rocky intertidal region (e.g., Menge 1976; Lubchenco and Menge 1978). Contrary to Lubchenco's and my conclusions, Petraitis concluded that (1) barnacles and not mussels are the favored prey of dogwhelks and (2) barnacles and not dogwhelks control mussel abundances in the mid and low rocky intertidal region. I provide evidence that these criticisms are unwarranted. First, Lubchenco and I never claimed that diet composition reflected prey preference. Moreover, predators can influence prey abundance without preferring the prey. Hence, claims regarding preferences have no bearing on our conclusions. Second, Petraitis' experiments do not invalidate Lubchenco's and my experimental results. Reanalyses of our experimental data support the earlier conclusion that at wave-sheltered sites, whelks reduce the abundance of mussels independently of barnacle abundances. Further, at all but one of Lubchenco's and my study sites, predator densities were higher than at Petraitis' site. Thus, the absence of a predator effect in Petraitis's study was most likely due to low predator density rather than a lack of generality of our earlier results. This reevaluation therefore suggests that within a broader conceptual framework, Petraitis' apparently divergent results are actually consistent with ours.     

Binding of polonium-210 to liver metallothionein

The interactions of 210Po at the molecular level in biological systems have received little study even though this alpha-emitting radionuclide occurs widely in nature. Polonium-210 was given subcutaneously to rats and found to be incorporated into liver metallothionein as judged by a number of criteria including heat stability, acetone precipitation, and chromatography. In vitro studies confirmed this binding. The binding of 210Po to metallothionein has implications that may help explain some of the radiation damage 210Po causes intracellularly.     

Studies on the genus Fontinalis (Musci: Fontinalaceae)

Fontinalis welchiana, a new species in the concave-leaved group ofFontinalis restricted to Arkansas, Missouri, and Illinois, differs fromF. novaeangliae by its slender size, dimorphic leaves, erect and strongly concave branch leaves, and lightly papillose exostome with ventral lamellae that are strongly rounded at the corners.Fontinalis allenii is a synonym ofF. antipyretica var.oreganensis.     

Montane plant communities of Fynbos Biome

The vegetation of the mountains of the Fynbos Biome is classified and described, mostly on the basis of vegetation structure and higher taxa. Various gradients can be recognized. A gradient in soil conditions runs from coarse textured, nutrient-poor soils in non-grassy fynbos (Mountain Fynbos) through grassy fynbos and grassy non-fynbos (Eastern Fynbos and Grasslands & Grassy Shrubland) to finer textured and less nutrient-poor soils in the non-fynbos Karroid & Renoster Shrubland. Another gradient of significance can be likened to the tropical gradient running from the dry conditions of hot semi-deserts to savannas or grasslands to woodlands. At the dry extreme an open shrubland occurs (Karroid & Renoster Shrubland, Asteraceous Fynbos), at intermediate positions a herbland occurs (Restioid Fynbos) and at the wet extreme another type of shrubland occurs (Ericaceous Fynbos). This kind of moisture gradient occurs on individual mountains with the xeric end being at the base of the north slopes, and the mesic end being on the upper south aspects. Soil characteristics are closely linked to this gradient; the better developed soils being found on the south aspects. Another moisture gradient is apparent in the vegetation. This gradient runs from the mesic southern coastal mountains to the north west where summer droughts are extremely severe although total rainfall may be similar.Nomenclature follows that used in the Government Herbarium, Stellenbosch.I thank William Bond, Richard Cowling, John Rushworth, Eddy van der Maarel and Marinus Werger for critical comments and discussion.     

Interaction of radiation-generated radicals with myoglobin in aqueous solution. III. Effects of oxygen and catalase on the product distribution in solutions of ferrimyoglobin containing N2O

The gamma-radiolysis of aqueous solutions of ferrimyoglobin in the presence of N2O at pH 7.3 has been examined as a function of added catalase and oxygen. Changes in the nature of the heme group have been monitored by visible absorption spectrophotometry and analysed quantitatively by a multiple wavelength method based on Beer's Law. Simple chemical analyses have been used to confirm qualitative identification of the product derivatives. As observed previously, the ferriheme is reduced by indirect globin-mediated action initiated by OH/H. The yield of reduced product decreases as [O2] increases. Conversion to ferrimyoglobin through the participation of H2O2 derived from irradiated water and from protein-mediated processes in oxygenated solution, is eliminated by the presence of catalase. Formation of a hemichrome form of ferrimyoglobin is apparent at higher doses in the presence of O2. These results demonstrate that oxygen plays an important role in controlling the nature and extent of redox that manifests ultimately on the heme group of ferrimyoglobin as a result of the initial interaction of OH/H.     

Nonrandom catabolism of proteins in the formation of antigenic peptide fragments

To examine the role of protein catabolism in the formation of antigenic peptide fragments, human fibrinopeptide-immune guinea pig T cells were stimulated with the large native molecule, human fibrinogen. Two different systems were tested. In the first, we determined responses by human fibrinopeptide B (hFPB)-immune T cells, to which strain (St.) 2 guinea pigs are responders and St. 13 are nonresponders, and by human fibrinopeptide A (hFPA)-immune T cells to which St. 13 are responders and St. 2 are nonresponders. Of interest in this comparison is that both hFPA and hFPB are amino terminal peptides on the A and B chain of fibrinogen, respectively, and are readily cleaved by thrombin during fibrin formation and by other trypsin-like enzymes, leaving a carboxyl terminal Arg. Thus, if fibrinogen catabolism occurred, both antigenic peptides should be equally represented for availability in T cell responses. It was found that hFPB-immune St. 2 T cells responded to fibrinogen, but no response was observed with hPFA-immune St. 13 T cells cultured with fibrinogen. To rule out that there was a general catabolic defect in St. 13 antigen-presenting cells, fibrinogen was presented by (2 X 13)F1 macrophages to fibrinopeptide-immune parental T cells. Again it was found that F1 macrophages could present fibrinogen to hFPB-immune T cells but failed to present hFPA. In another comparison, responses with fibrinogen were also determined with des-ARg-hFPB, which lacks the carboxyl terminal Arg of hFPB, to which St. 13 are responders and St. 2 are nonresponders. The advantage of this comparison is that both antigenic determinants are contained within the same small peptide. St. 13 des-Arg-hFPB-immune T cells failed to respond in vitro by culture with human fibrinogen, suggesting that these antigenic determinants are not produced from larger peptides or proteins containing those determinants. To rule out the possibility that this was only an in vitro phenomenon, guinea pigs were immunized with the larger protein, the B chain of fibrinogen, and the immune T cells were examined for responses to fibrinopeptides derived from the B chain. Immune St. 2 T cells responded to hFPB but not to des-Arg-hFPB, whereas St. 13 T cells remained unresponsive with both peptides. These results indicate that proteolysis of larger proteins to form small antigenic peptides is not a random event and that not all potential antigenic determinants contained in a protein are produced during antigen processing.