Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

Further characterization of human erythrocyte superoxide dismutase.

1. A simplified procedure for the preparation of highly purified human superoxide dismutase from erythrocytes was developed which avoided extremes of pH and ionic strength and the use of organic solvents; the properties of human and bovine proteins, prepared by the method, were compared. 2. Using the two dimensional electrophoretic procedure of O'Farrell, the human superoxide dismutase was found to consist of a single type of polypeptide. 3. The human protein was found to have a total of eight half-cystine residues per mole of protein, compared to six such residues for the bovine protein. The human protein has two sulfhydryl groups which are reactive toward mercurials when dissolved in 1M guanidine-hydrochloride and approximately 3 reactive sulfhydrls when the protein is dissolved in 6 M guanidine hydrochloride. The distribution of the eight sulfur atoms appears to consist of four involved in disulfide linkages, two deeply buried within the molecule and unreactive except under strongly denaturing conditions, and two which are reactive under mildly denaturing conditions. No zero-valent sulfur was found. 4. The visible optical absorption, the visible circular dichroism, and the electron paramagnetic resonance spectra are essentially identical with those of the bovine protein. No unusual absorbance was found at 330 nm. The near ultraviolet spectrum is different from that of the bovine protein, and this appears to be due to differing amino acid compositions. 5. Two fractions of superoxide dismutase activity were observed during chromatography of partially purified solutions on diethylaminoethyl-cellulose. The minor, less mobile form, was found to revert to the less mobile species on aging; the reverse process was not observed to occur. The minor component was found to contain equimolar amounts of Zn and Cu and to have a specific dismutase activity somewhat higher than that of the purified major fraction.     

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.     

Experimental studies on a lethal gene (1) in the Mexican axolotl, Ambystoma mexicanum.

1. Gene ? is a recessive lethal factor found in the white strain of axolotls. Animals heterozygous for the gene are phenotypically normal. When mated with each other they give offspring 25% of which exhibit the lethal effects of the gene. 2. The ?/? homozygotes develop normally to an advanced embryonic stage (Harrison stage 40) before the effects of the gene are first manifested. They then come to display a characteristic combination of abnormalities, including a disproportionately small head, small and poorly developed eyes, abnormal poorly developed gills, undifferentiated limb buds, and reduced overall growth rate. They may feed briefly, but soon stop and invariably die within a few weeks of the time of hatching. 3. The action of gene ? has been analyzed by parabiosing mutant and normal embryos, and by grafting various organ primordia reciprocally between mutant and normal embryos. Parabiosis to normal embryos fails to correct the abnormalities of the mutants, although their survival may be somewhat prolonged. Grafts of mutant organ primordia (eye, limb, gill, pronephros, gonad, head) also invariably fail to show improved development or to survive on normal hosts; normal organ primordia develop normally on mutant hosts so long as the mutant survives. These experiments indicate that gene ? is a recessive autonomous cell lethal affecting all of the organ systems during late embryonic and early larval development.     

Primary structure of the nucleotide binding domain of the Ca,Mg-ATPase from cardiac sarcoplasmic reticulum

The nucleotide binding domain of the active site of the Ca,Mg-ATPase of cardiac sarcoplasmic reticulum (SR) has been isolated using fluorescein isothiocyanate (FITC) as an active site label and sequenced. After removal of non-specifically incorporated FITC with hydroxylamine, the amount of label incorporated was stoichiometric with residual ATPase activity, demonstrating that the label was incorporated uniquely at the active site. The SR was succinylated before digestion by trypsin in order to obtain a peptide of sufficient length to determine if the cardiac SR ATPase is a candidate for the unidentified cDNA clone recently sequenced by MacLennan et al. (Nature 316: 696-700, 1985). The sequence of the labeled SR peptide, obtained by affinity chromatography on a FITC antibody column, was T S M S K M F K G P E V I D R. This sequence was identical with that predicted by the unidentified clone and is significantly different from the sequence reported by Kirley et al. (Biochem. Biophys. Res. Commun. 130: 732-738, 1985) for a FITC labeled peptide isolated from cardiac SR.     

Disease hotspots or hot species? Infection dynamics in multi‐host metacommunities controlled by species identity,not source location

Pathogen persistence in host communities is influenced by processes operating at the individual host to landscape‐level scale, but isolating the relative contributions of these processes is challenging. We developed theory to partition the influence of host species, habitat patches and landscape connectivity on pathogen persistence within metacommunities of hosts and pathogens. We used this framework to quantify the contributions of host species composition and habitat patch identity on the persistence of an amphibian pathogen across the landscape. By sampling over 11 000 hosts of six amphibian species, we found that a single host species could maintain the pathogen in 91% of observed metacommunities. Moreover, this dominant maintenance species contributed, on average, twice as much to landscape‐level pathogen persistence compared to the most influential source patch in a metacommunity. Our analysis demonstrates substantial inequality in how species and patches contribute to pathogen persistence, with important implications for targeted disease management.     

Incidence of lamprey marks on Lake Sturgeon (Acipenser fulvescens Rafinesque, 1817) in the St. Clair – Detroit River System: Implications for Sea Lamprey (Petromyzon marinus Linnaeus, 1758) effects

Laurentian Great Lakes Lake Sturgeon (Acipenser fulvescens) are hosts to lamprey species, including native Silver Lamprey (Ichthyomyzon unicuspis) and invasive Sea Lamprey (Petromyzon marinus). Silver Lamprey coevolved with Lake Sturgeon and cause negligible mortality, but Sea Lamprey can negatively affect Lake Sturgeon populations. Sea Lamprey abundance in Lake Erie has been above targets set by resource managers, with the St. Clair – Detroit River System (SCDRS) suspected as a source of Sea Lamprey production into Lake Erie. This study summarizes lamprey marking on Lake Sturgeon captured during agency assessment surveys in the SCDRS since 1996 and provides insight on the potential for Sea Lamprey to negatively affect Lake Sturgeon in the SCDRS. Lamprey marks (any lamprey species) were noted on 48.2% of Lake Sturgeon (2.5 marks/fish) and 3.3% of Lake Sturgeon assumed to be susceptible to mortality by Sea Lamprey (<760 mm TL; 0.06 marks/fish). Silver Lamprey were the only lamprey species found attached to Lake Sturgeon and there was no difference between oral disc diameters of Silver Lamprey and marks measured on Lake Sturgeon in Lake St. Clair and the lower St. Clair River (p = .45). Based on logistic regression, probability of at least one lamprey mark increased with Lake Sturgeon total length and was highest in Lake St. Clair. The probability of observing at least one lamprey mark on a 760 mm Lake Sturgeon was 8.1% or less for each sampling location in the SCDRS aside from Lake St. Clair (28.1%). Results suggest that parasitism of Lake Sturgeon by Sea Lamprey in the SCDRS is rare, particularly for Lake Sturgeon <760 mm TL. Low incidence of lamprey marks on Lake Sturgeon assumed to be susceptible to mortality from Sea Lamprey parasitism and zero occurrence of Sea Lamprey being observed attached to a Lake Sturgeon suggest Sea Lamprey at their current abundance likely have little effect on the Lake Sturgeon population in the SCDRS. Caution should be taken when using mark size to assign marks to lamprey species as there is substantial overlap among species oral disc diameters, potentially inflating the perceived impact of Sea Lamprey on Lake Sturgeon in areas with native lampreys.     

Legacies of Prehistoric Agricultural Practices Within Plant and Soil Properties Across an Arid Ecosystem

Closely integrated research between archaeologists and ecologists provides a long-term view of human land use that is rare in the ecological literature, allowing for investigation of activities that lead to enduring environmental outcomes. This extended temporal perspective is particularly important in aridlands where succession occurs slowly and ecosystem processes are mediated by abiotic, geomorphic factors. Numerous studies show that impacts from ancient human actions can persist, but few have explored the types of practices or mechanisms that lead to either transient or long-term environmental change. We compared plant and soil properties and processes from a range of landscape patch types in the Sonoran Desert of the US Southwest that supported different, well-documented prehistoric farming practices from AD 750–1300. Our results show that the types of ancient human activities that leave long-term ecological legacies in aridlands are those that fundamentally alter “slow variables” such as soil properties that regulate the timing and supply of water. Prehistoric Hohokam floodwater-irrigation practices, but not dryland farming techniques, substantially altered soil texture, which was strongly associated with desert plant community and functional composition. However, prehistoric agriculture did not consistently alter long-term nutrient availability and thus had no impact on “fast variables” such as production of seasonal annual plants that are restricted to periods of ample rainfall. In this arid ecosystem, the inverse texture model explained patterns in plant functional composition at large scales, but is less predictive of production of short-lived desert annuals that experience a more mesic precipitation regime.