Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

The chronic lymphatic leukemia lymphocyte: Correlation of functional,metabolic, and surface immunoglobulin characteristics

This study determined the correlation between the functional capacity of chronic lymphatic leukemia lymphocytes as determined by their response to nonspecific mitogens with their glucose metabolism and surface immunoglobulin characteristics. A majority of patients (12) were found to have lymphocytes with impaired transformation to both PHA and pokeweed mitogens. These cells also had impaired glucose metabolism in unstimulated cultures and failed to have the striking increase in glucose metabolism in response to mitogens which is characteristic of normal lymphocytes. Most of these lymphocytes had IgM surface immunoglobulins. However, we were not able to demonstrate surface immunoglobulins on the lymphocytes of one patient in this group. Two patients were found to have lymphocytes with normal lymphoblastic transformation to PHA and impaired transformation to pokeweed suggesting cells of T origin. The glucose metabolism of these lymphocytes were less impaired in unstimulated cultures than those of the other patients and had a striking increment in glucose metabolism in response to PHA similar to normal lymphocytes. Unexpectedly, these lymphocytes were found to have IgG on their surface suggesting cells of B origin. These results indicate that there may be two groups of CLL patients with clinically similar disease in whom the functional and metabolic characteristics of the lymphocytes are distinct and that the surface immunoglobulin characteristic of lymphocytes may not always predict their functional characteristic.     

Localization in the cell cycle of the antimitotic action of the pyrrolizidine alkaloid,lasiocarpine and of its metabolite,dehydroheliotridine

The antimitotic action of the pyrrolizidine alkaloid lasiocarpine on rat liver parenchyma was investigated using as the experimental model the wave of mitosis produced in liver by a single dose of thioacetamide. A single low dose of lasiocarpine administered two weeks before the thioacetamide, almost completely inhibited the mitotic wave without inhibiting to the same extent the preceding wave of DNA synthesis. By the use of selective inhibitors and radioisotope labelling, the location of the mitotic block was found to be either in the latter half of the DNA synthetic phase, S, or early in G₂, the post-synthetic phase. The mitotic wave was similarly inhibited by pretreatment of the rats with a single injection of dehydroheliotridine, a pyrrolic metabolite of heliotridine-based pyrrolizidine alkaloids.     

Fluorescent-Antibody Study of Natural Finger-Like Zooloeae

Fluorescent-antibody techniques using Zoogloea ramigera 106 antiserum were used to study fresh activated sludge flocs and finger-like zoogloeae in the microbial film that developed over stored samples of activated sludge. Few cells in fresh activated sludge reacted positively with the fluorescein-labeled antiserum. Finger-like zoogloeae containing reactive cells were readily observed in the microbial film layer over stored activated sludge. Certain of the natural finger-like projections were entirely composed of cells that reacted positively to the labeled Z. ramigera 106 antiserum, whereas other projections were devoid of reactive cells.     

Proteolytic activity within lysosomes and turnover of pinocytic vesicles a kinetic analysis

In previous studies, in vitro digestion of [^{1 2 5}I] ribonuclease by lysosomes of mouse kidney was limited because breakdown, which was rapid at first slowed markedly so that most of the labeled protein escaped degradation. We now describe incubation conditions which allow digestion to proceed until approximately 70% of the exogenous protein label is released in acid-soluble from, after 30–45 min at 37°C. Such activity is seen with either the addition of EDTA or incubation of concentrated cell particle suspensions. EDTA is effective in low concentrations and shows the same stimulation of digestion over a range of approximately 10⁻⁶−10⁻³ M. Other chelating agents have similar effects; dipyridyl and hydroxquinoline are as effective as EDTA, o-phenanthroline and diethyldithiocarbamate are slightly less effective. When the incubation medium had been treated with a chelating resin, Chelex 100, dilute suspensions of lysosomes were as active as those in EDTA. These results lead to the conclusion that metal ions, present as contaminants in very small concentraions, inhibit the activity of mouse kidney lysosomes.The effect of the metal ions is to diminish lysosomal stability, leading to release of intact labeled ribonuclease in non-sedimentable form. Interaction between lysosomes and metal, leading to inhibition of digestion upon heating occurs at low temperature, but breakdown requires incubation at 37°C and may be autolytic. In contrast to chelators, mercaptoethanol is without marked effect on stability; the stimulation in digestion rate caused by this agent is due either to a direct effect on the lysosomal enzymes or to a non-destructive influence on the lysosomal structure.     

Informing conservation by identifying range shift patterns across breeding habitats and migration strategies

A species distribution combines the resources and climatic tolerances that allow an individual or population to persist. As these conditions change, one mechanism to maintain favorable resources is for an organism to shift its range. Much of the research examining range shifts has focused on dynamic distribution boundaries wheras the role of species breeding habitat or migration strategies on shift tendencies has received less attention. We expand on previous research by using a large suite of avian species (i.e., 277), analyzing observed abundance-weighted average latitudes, and categorizing species by breeding environment and migration strategy. We used the North American Breeding Bird Survey dataset to address two questions: (1) Has the center of observed abundance for individual species shifted latitudinally? (2) Is there a relationship between migration strategy or breeding habitat and range shifts? Results indicate the majority of species have experienced poleward range shifts over the last 43 years, and birds breeding in all habitat showed trends of poleward shift but only those species breeding in scrub-shrub and grassland environments were different from zero. Additionally, species that are short distance migrants are experiencing significant poleward shifts while Neotropical and permanent residents had shifts that were not different from zero. Our findings do support the general trend expected from climate driven changes (i.e., > 52 % shifting poleward), however, the proportion of species exhibiting equatorial shifts (24 %) or no significant shifts (23 %) illustrates the complex interplay between land cover, climate, species interactions, and other forces that can interact to influence breeding ranges over time. Regardless of the mechanisms driving range shifts, our findings emphasize the need for connecting and expanding habitats for those species experiencing range shifts. This research describes the patterns of breeding birds through central North America and we encourage future research to focus on the mechanisms driving these patterns.     

New evidence for hybrid zones of forest and savanna elephants in Central and West Africa

The African elephant consists of forest and savanna subspecies. Both subspecies are highly endangered due to severe poaching and habitat loss, and knowledge of their population structure is vital to their conservation. Previous studies have demonstrated marked genetic and morphological differences between forest and savanna elephants, and despite extensive sampling, genetic evidence of hybridization between them has been restricted largely to a few hybrids in the Garamba region of northeastern Democratic Republic of Congo (DRC). Here, we present new genetic data on hybridization from previously unsampled areas of Africa. Novel statistical methods applied to these data identify 46 hybrid samples – many more than have been previously identified – only two of which are from the Garamba region. The remaining 44 are from three other geographically distinct locations: a major hybrid zone along the border of the DRC and Uganda, a second potential hybrid zone in Central African Republic and a smaller fraction of hybrids in the Pendjari–Arli complex of West Africa. Most of the hybrids show evidence of interbreeding over more than one generation, demonstrating that hybrids are fertile. Mitochondrial and Y chromosome data demonstrate that the hybridization is bidirectional, involving males and females from both subspecies. We hypothesize that the hybrid zones may have been facilitated by poaching and habitat modification. The localized geography and rarity of hybrid zones, their possible facilitation from human pressures, and the high divergence and genetic distinctness of forest and savanna elephants throughout their ranges, are consistent with calls for separate species classification.     

Loss of Starch Granule Initiation Has a Deleterious Effect on the Growth of Arabidopsis Plants Due to an Accumulation of ADP-Glucose

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.The metabolism of starch plays an essential role in the physiology of plants. Starch breakdown provides the plant with carbon skeletons and energy when the photosynthetic machinery is inactive (transitory starch) or in the processes of germination and sprouting (storage starch). Deficiencies in the accumulation of transitory starch in Arabidopsis (Arabidopsis thaliana) have been described previously, specifically in mutants affected in the plastidial phosphoglucomutase (PGM1) or the small subunit (APS1) of the ADP-Glc pyrophosphorylase (AGPase). While they are described as “starchless,” they actually contain small amounts of starch (1%–2% of the wild-type levels; Streb et al., 2009) and share similar phenotypic alterations, such as growth retardation when cultivated under a short-day photoregime and increased levels of soluble sugars during the light phase and reduced levels during the night (Caspar et al., 1985; Lin et al., 1988b; Schulze et al., 1991). Carbon partitioning is altered in these plants. As photosynthate cannot be accumulated as starch, it is diverted via hexose phosphates in the cytosol to the synthesis of Suc, which accumulates together with the hexose sugars, Glc and Fru (Caspar et al., 1985). In Arabidopsis, there are five starch synthase isoforms: one granule-bound starch synthase and four soluble starch synthases: SS1, SS2, SS3, and SS4. We have described previously an Arabidopsis mutant plant lacking SS3 and SS4 that is also severely affected in the accumulation of starch (Szydlowski et al., 2009). SS4 is involved in the initiation of the starch granule and controls the number of granules per chloroplast (Roldán et al., 2007). The elimination of SS3 in an ss4 background leads to an absence of starch in most of the chloroplasts, despite the fact that SS1 and SS2 are still present and total starch synthase activity is only reduced by 35% (Szydlowski et al., 2009). However, a very small proportion of chloroplasts of this mutant plant contain a single huge starch granule, which is also a characteristic of chloroplasts in the ss4 single mutant (D’Hulst and Mérida, 2012). Thus, like aps1 and pgm1, ss3/ss4 plants contain only small amounts of starch. However, unlike aps1 or pgm1 plants, most of the cells of this mutant have empty chloroplasts, without starch (Szydlowski et al., 2009).In this work, we have analyzed the phenotypic effects of the impaired starch accumulation of ss3/ss4 plants. We show that this mutant displays phenotypic changes that are not found in other mutants with very low levels of starch, such as aps1 or pgm1 plants. We provide evidence that extremely high levels of ADP-Glc accumulate in the ss3/ss4 plants. Using reverse genetics to block the pathway of starch synthesis upstream of the starch synthases reduced the level of ADP-Glc in ss3/ss4 plants and reverted the other phenotypic traits. This suggests that ADP-Glc accumulation is the causal factor behind the chlorotic and stunted growth phenotypes of the ss3/ss4 mutant.     

Wheat alleles introgress into selfing wild relatives: empirical estimates from approximate Bayesian computation in Aegilops triuncialis

Extensive gene flow between wheat (Triticum sp.) and several wild relatives of the genus Aegilops has recently been detected despite notoriously high levels of selfing in these species. Here, we assess and model the spread of wheat alleles into natural populations of the barbed goatgrass (Aegilops triuncialis), a wild wheat relative prevailing in the Mediterranean flora. Our sampling, based on an extensive survey of 31 Ae. triuncialis populations collected along a 60 km × 20 km area in southern Spain (Grazalema Mountain chain, Andalousia, totalling 458 specimens), is completed with 33 wheat cultivars representative of the European domesticated pool. All specimens were genotyped with amplified fragment length polymorphism with the aim of estimating wheat admixture levels in Ae. triuncialis populations. This survey first confirmed extensive hybridization and backcrossing of wheat into the wild species. We then used explicit modelling of populations and approximate Bayesian computation to estimate the selfing rate of Ae. triuncialis along with the magnitude, the tempo and the geographical distance over which wheat alleles introgress into Ae. triuncialis populations. These simulations confirmed that extensive introgression of wheat alleles (2.7 × 10^?4 wheat immigrants for each Ae. triuncialis resident, at each generation) into Ae. triuncialis occurs despite a high selfing rate (Fis ≈ 1 and selfing rate = 97%). These results are discussed in the light of risks associated with the release of genetically modified wheat cultivars in Mediterranean agrosystems.