Soybean ferritin: isolation, characterization, and free radical generation

The main aim of this work was to assess the multi-task role of ferritin(Ft)in the oxidative metabolism of soybean(Glycine max).Soybean seeds incubated for 24 h yielded 41 ± 5 μg Ft/g fresh weight.The rate of in vitro incorporation of iron(Fe)into Ft was tested by supplementing the reaction medium with physiological Fe chelators.The control rate,observed in the presence of 100 μM Fe,was not significantly different from the values observed in the presence of 100 μM Fe-his.However,it was significantly higher in the presence of 100 μM Fe-citrate(approximately 4.5-fold)or of 100 μM Fe-ATP(approximately 14-fold).Moreover,a substantial decrease in the Trp-dependent fluorescence of the Ft protein was determined during Fe uptake from Fe-citrate,as compared with the control.On the other hand,Ft addition to homogenates from soybean embryonic axes reduced endogenously generated ascorbyl radical,according to its capacity for Fe uptake.The data presented here suggest that Ft could be involved in the generation of free radicals,such as hydroxyl radical,by Fe-catalyzed reactions.Moreover,the scavenging of these radicals by Ft itself could then lead to protein damage.However,Ft could also prevent cellular damage by the uptake of catalytically active Fe.     

Disentangling the paradox of insect phenology: are temporal trends reflecting the response to warming?

The strength and direction of phenological responses to changes in climate have been shown to vary significantly both among species and among populations of a species, with the overall patterns not fully resolved. Here, we studied the temporal and spatial variability associated with the response of several insect species to recent global warming. We use hierarchical models within a model comparison framework to analyze phenological data gathered over 40 years by the Japan Meteorological Agency on the emergence dates of 14 insect species at sites across Japan. Contrary to what has been predicted with global warming, temporal trends of annual emergence showed a later emergence day for some species and sites over time, even though temperatures are warming. However, when emergence data were analyzed as a function of temperature and precipitation, the overall response pointed out an earlier emergence day with warmer conditions. The apparent contradiction between the response to temperature and trends over time indicates that other factors, such as declining populations, may be affecting the date phenological events are being recorded. Overall, the responses by insects were weaker than those found for plants in previous work over the same time period in these ecosystems, suggesting the potential for ecological mismatches with deleterious effects for both suites of species. And although temperature may be the major driver of species phenology, we should be cautious when analyzing phenological datasets as many other factors may also be contributing to the variability in phenology.     

Kin recognition by paternal half-siblings in captive Papio cynocephalus

Our objective in this study was to evaluate whether a group of paternally related, subadult baboons (Papio cynocephalus) would preferentially interact with kin or nonkin when they had been raised apart from kin other than their mothers. Subjects and their mothers were removed from the breeding group and placed in alternate housing within 24 h after birth to ensure that the subjects would not have a social history with either their sire or their half-siblings. At 90 days of age, the 23 subjects were separated from their mothers and assigned to a peer–peer social group. Behavioral performance was measured using focal animal sampling techniques and 12 molecular behavioral criteria. Analyses of the data indicate that in dyadic interactions kin did not interact more frequently than nonkin in performance of affiliative, sociosexual, and agonistic behaviors. The hypothesis that baboons recognize kin in the absence of maternal associations was not supported by the data; moreover, we suggest that social learning and social history are the most likely mechanisms for kin recognition. Am. J. Primatol. 43:147–157, 1997. © 1997 Wiley-Liss, Inc.     

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.     

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.     

Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response

The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms. In an effort to identify novel proteins that might be involved in stabilizing stalled replication forks, Saccharomyces cerevisiae mutant wss1Δ was isolated from a high-throughput screening of ~5000 deletion strains for genes involved in the response to continuous, low-intensity UV irradiation. Disruption of WSS1 resulted in synergistic increases in UV sensitivity with null mutants of genes involved in recombination (RAD52) and cell cycle control (RAD9 and RAD24). WSS1 was also found to interact genetically with SGS1, TOP3, SRS2 and CTF4, which are involved in recombination, repair of replication forks and the establishment of sister chromatid cohesion. A yeast two-hybrid screen identified a potential physical interaction between Wss1 and both Psy2 and Tof1. Genetic interactions were also detected between PSY2 and TOF1, as well as between each gene and RAD52 and SRS2, and between WSS1 and TOF1. Tof1 is known to be involved in stabilizing stalled replication forks and our data suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks.     

Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans.