Chloroplast-targeted ERD1 protein declines but its mRNA increases during senescence in Arabidopsis

Arabidopsis ERD1 is a ClpC-like protein that sequence analysis suggests may interact with the chloroplast-localized ClpP protease to facilitate proteolysis. The mRNA encoded by the ERD1 gene has previously been shown to accumulate in response to senescence and to a variety of stresses and hormones. Here we show that the ERD1 protein, in contrast to the ERD1 mRNA, strongly declines in abundance with age, becoming undetectable in fully expanded leaves. Sequence analysis also suggests that ERD1 is chloroplast targeted, and we show in an in vitro system that the native protein is properly imported, processed, and present within the soluble fraction of the chloroplast, presumably the stroma. We show that ClpP protein, which is also present in the stroma, declines with age in parallel with ERD1. These results are consistent with the interaction of ERD1 and ClpP, but they suggest that it is unlikely that either plays a major role during senescence. Certain other chloroplast proteins decline with age coordinately with ERD1 and ClpP, suggesting that these declines are markers of an early age-mediated change that occurs within the chloroplast.     

The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C- domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so- called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.     

An unanticipated source of hope: stigma and cervical cancer in Brazil

In this article, I argue that although cervical cancer is an often stigmatized condition in Brazil, women with cervical cancer in Recife, Brazil, did not simply endure the stigma, they also perpetuated it. I draw on narrative theory and 18 months of ethnographic research in Recife to argue that rather than resisting the stigma associated with their disease, women in Recife used stigma to construct illness narratives that affirmed that they were still held to the same norms and values as the nonill. In turn, those narratives, and the healing narratives constructed along with them, provided women with hope for a future free from cervical cancer and free from the "imperfections" associated with that disease. Thus, women with cervical cancer used stigmatizing narratives both as links back to the "normal" world they inhabited before they became ill, and as bridges forward to the future they hoped to attain.     

Calling is an honest indicator of paternal genetic quality in poison frogs

Several competing hypotheses have been put forward to explain why females of many species mate preferentially with males possessing the most conspicuous signals (e.g., ornaments, displays, or songs). We performed a laboratory experiment using two species of poison frogs, Dendrobates leucomelas and Epipedobates tricolor, to test the hypothesis that male calling performance is an honest indicator of parental quality. Our analyses are based on data from behavioral observations of mating activities of captive-reared individuals (and their offspring) that were housed in terraria for four consecutive breeding seasons. Male mating success increased with male calling rate and chirp duration in both species, suggesting that females preferred males with more elaborate calls. Because calling performance improved with age in D. leucomelas, female poison frogs that prefer males with more elaborate calls in the wild may end up mating with older males that have already proven their ability to survive. Females that mated with good callers obtained higher quality offspring. Eggs fertilized by males with high calling rates and long chirp durations had higher hatching success and produced tadpoles that were more likely to metamorphose into surviving frogs. As a consequence, females that mated with males with high calling performance obtained more surviving offspring per egg, compared to females that mated with poor callers. Collectively, our findings comply with the notion that female poison frogs prefer to mate with good callers because calling performance is a reliable predictor of offspring quality. The possible influence of maternal allocation and reasons for the strong effect size compared to previous studies are discussed.     

汶川地震对大熊猫主食竹——拐棍竹竹笋生长发育的影响

为了了解汶川地震对大熊猫主食竹生长发育的影响,2009年10月—2011年4月,在地震重灾区四川龙溪-虹口国家级自然保护区内设置150个样方,就地震强度、中度、轻度干扰对大熊猫主食竹拐棍竹(Fargesia robusta)竹笋生长发育的影响进行了研究。研究结果显示:(1)发笋物候上,地震强度干扰的发笋时间(Julian时间)较中度和轻度干扰显著推迟,而发笋期不受影响。(2)发笋量和当年生幼竹数是拐棍竹更新生长的数量指标。地震强度和中度干扰样方的发笋量都显著高于轻度干扰,而发笋量在强度和中度干扰间差异不显著;地震强度干扰样方的当年生幼竹数显著高于中度和轻度干扰,中度和轻度干扰样方间差异不显著。这说明地震的强度和中度干扰都有利于拐棍竹竹笋和当年生幼竹数量的更新。(3)基径和株高是拐棍竹更新生长的质量指标。强度干扰下拐棍竹的基径和株高都显著小于中度和轻度干扰,中度干扰样方的基径与轻度干扰差异不显著,但株高显著矮于轻度干扰样方。表明强度干扰下拐棍竹的质量最差,中度干扰的基径已基本得到恢复,但株高仍受到抑制。(4)地震不同干扰等级拐棍竹退笋原因划分为:外力机械损伤、动物取食、生长停止、其他4种。对地震不同干扰等级拐棍竹退笋原因比率的分析发现,强度干扰样方因外力机械损伤和生长停止退笋的比率显著高于中度和轻度干扰样方,而因动物取食退笋的比率显著低于中度和轻度干扰样方。证明强度干扰样方的竹笋更容易受到外力机械损伤和生长停止退笋,而中度和轻度干扰的竹笋更倾向于动物取食致退,揭示了拐棍竹的生长是与震后不同程度的干扰相适应的。     

Halide peroxidase in tissues that interact with bacteria in the host squid Euprymna scolopes

An enzyme with similarities to myeloperoxidase, the antimicrobial halide peroxidase in mammalian neutrophils, occurs abundantly in the light organ tissue of Euprymna scolopes, a squid that maintains a beneficial association with the luminous bacterium Vibrio fischeri. Using three independent assays typically applied to the analysis of halide peroxidase enzymes, we directly compared the activity of the squid enzyme with that of human myeloperoxidase. One of these methods, the diethanolamine assay, confirmed that the squid peroxidase requires halide ions for its activity. The identification of a halide peroxidase in a cooperative bacterial association suggested that this type of enzyme can function not only to control pathogens, but also to modulate the interactions of host animals with their beneficial partners. To determine whether the squid peroxidase functions under both circumstances, we examined its distribution in a variety of host tissues, including those that typically interact with bacteria and those that do not. Tissues interacting with bacteria included those that have specific cooperative associations with bacteria (i.e., the light organ and accessory nidamental gland) and those that have transient nonspecific interactions with bacteria (i.e., the gills, which clear the cephalopod circulatory system of invading microorganisms). These bacteria-associated tissues were compared with the eye, digestive gland, white body, and ink-producing tissues, which do not typically interact directly with bacteria. Peroxidase enzyme assays, immunocytochemical localization, and DNA-RNA hybridizations showed that the halide-dependent peroxidase is consistently expressed in high concentration in tissues that interact bacteria. Elevated levels of the peroxidase were also found in the ink-producing tissues, which are known to have enzymatic pathways associated with antimicrobial activity. Taken together, these data suggest that the host uses a common biochemical response to the variety of types of associations that it forms with microorganisms.     

Different modes of interaction by TIAR and HuR with target RNA and DNA

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.     

Bacteraemia in free-ranging Hawaiian green turtles Chelonia mydas with fibropapillomatosis

Past studies of free-ranging green turtles Chelonia mydas with fibropapillomatosis (FP) in Hawaii have shown that animals become immunosuppressed with increasing severity of this disease. Additionally, preliminary clinical examination of moribund turtles with FP revealed that some animals were also bacteraemic. We tested the hypothesis that bacteraemia in sea turtles is associated with the severity of FP. We captured free-ranging green turtles from areas in Hawaii where FP is absent, and areas where FP has been endemic since the late 1950s. Each turtle was given an FP severity score ranging from 0 (no tumours) to 3 (severely affected). A fifth category included turtles that were stranded ashore and moribund with FP. We found that the percentage of turtles with bacteraemia increased with the severity of FP, and that the majority of bacteria cultured were Vibrio spp. Turtles with severe FP were more susceptible to bactaeremia, probably in part due to immunosuppression. The pattern of bacteraemia in relation to severity of disease strengthens the hypothesis that immunosuppression is a sequel to FP.     

Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus.

The BJ cell line which constitutively expresses herpes simplex virus 1 glycoprotein D is resistant to infection with herpes simplex viruses. Analysis of clonal lines indicated that resistance to superinfecting virus correlates with the expression of glycoprotein D. Resistance was not due to a failure of attachment to cells, since the superinfecting virus absorbed to the BJ cells. Electron microscopic studies showed that the virions are juxtaposed to coated pits and are then taken up into endocytic vesicles. The virus particles contained in the vesicles were in various stages of degradation. Viral DNA that reached the nucleus was present in fewer copies per BJ cell than that in the parental BHKtk- cells infected at the same multiplicity. Moreover, unlike the viral DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. The results suggest that the glycoprotein D expressed in the BJ cell line interfered with fusion of the virion envelope with the plasma membrane but not with the adsorption of the virus to cells and that the viral proteins that mediate adsorption to and fusion of membranes appear to be distinct.     

Evolution of a pulmonary insulin delivery system (Exubera) for patients with diabetes

Many patients with diabetes fail to meet recommended glycemic goals regardless of the recognition of optimal glycemic control as a key component for improving clinical outcomes and quality of life in patients with diabetes. Patient- and physician-related barriers to the adoption of insulin therapy include fear and anxiety about injecting insulin, concerns about side effects, and personal health beliefs in regard to the use of insulin. There is an unmet need for an alternative insulin therapy that provides optimal glycemic control, is well tolerated, and improves patient adherence. Of the several inhaled insulin devices that are in various stages of development, the Exubera (INH) formulation is the first to be approved for use in the United States and in Europe. Exubera is a novel, rapid-acting inhaled human insulin formulation that has been developed for prandial insulin use. Clinical studies have shown that INH consistently improves glycemic control, in combination with longer-acting subcutaneous (SC) insulin regimens in patients with type 1 or type 2 diabetes, or is used to supplement or replace oral antidiabetic therapy in patients with type 2 diabetes. INH has demonstrated long-term safety and tolerability, with a risk for hypoglycemia similar to that of SC insulin, and no clinically meaningful changes in pulmonary function have been noted with its use. Patients treated with INH in clinical studies reported high levels of satisfaction with treatment, and many patients with diabetes choose inhaled insulin when it is offered as a treatment option. Taken together, these findings suggest that INH represents an important new development in the treatment of diabetes that may improve glycemic control in many patients with diabetes.     

Triangular fecundity function and ageing in ladybird beetles

Abstract 1. In insects, the age schedules of fecundity tend to be triangular and this has been attributed more to temporal patterns of mortality than to fecundity. The objective of the work reported here was to test the assumption that senescence shapes the fecundity function in ladybird beetles, and in particular that the production function declines with age.
2. The results of a laboratory study on three species of predatory ladybird beetle indicated that the efficiency with which these insects acquire and process food declined with age. Although supplied with the same amount of food each day, after the onset of reproduction, these beetles ate less and less with increasing age. Egg production mirrored the decline in aphid consumption. Associated with this was a decline in fertility, assimilation, and speed of locomotion with age.
3. This study indicates that production declined with age and that this shaped the fecundity schedules in these ladybird beetles. In addition, the results indicated that ladybirds are income breeders and, as predicted, the reproductive effort of the small species was greater than that of the large species used in this study.     

Diffusion in the slice microenvironment and implications for physiological studies

The brain cell microenvironment includes the extracellular space surrounding the cell together with the cellular elements that define the space. The dense packing of cells in the mammalian nervous system ensures that the extracellular space is narrow but highly complex in geometry. Recent studies with ion-selective micropipettes have revealed that the cerebellar slice can support changes in [K+]o that resemble those seen in the intact preparation. In the slice, [K+]o responses of individual cells can even be resolved. Studies with iontophoretic techniques and quantitative analysis in the slice have shown that the extracellular space has diffusion properties, characterized by a volume fraction and a tortuosity, that are very similar to those seen in the intact animal. These data confirm that the microenvironment in the slice is comparable to that in the intact animal. The diffusion parameters can be used to make predictions about the time necessary for substances to diffuse into slices under various conditions. Such estimates, together with other studies, indicate that it is probably inadvisable to use slices with thicknesses in excess of 300--400 micrometers, and that the bathing conditions can be critical in maintaining slice viability.     

An invasion of cheats; the evolution of worthless nuptial gifts

Nuptial gifts are food items or inedible tokens that are transferred to females during courtship or copulation . Tokens are of no direct value to females, and it is unknown why females require such worthless gifts as a precondition of mating. One hypothesis is that token giving arose in species that gave nutritious gifts and males exploited female preferences for nutritional gifts by substituting more easily obtainable but worthless items. An invasion of such behavior would require that females accept the substitute gift and copulate for a period of time similar to that with genuine gifts. We show that both these prerequisites are met in the dance fly Rhamphomyia sulcata, in which females normally accept a nutritious gift. We removed the gift from copulating pairs and replaced it with either a large or small prey item or inedible token. We found that although pairs copulated longest with a large genuine gift, the tokens resulted in copula durations equivalent to those with a small genuine gift. We also observed that males that returned to the lek with tokens re-paired successfully. These findings suggest that female behavior in genuine gift-giving species is susceptible to the invasion of male cheating on reproductive investment.     

Subcellular localization of the human proto-oncogene protein DEK

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.     

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.     

The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycle

Summary The outer tangential wall (OTW) of epidermal cells of azuki bean epicotyls has a crossed polylamellate structure, in which lamellae of longitudinal cellulose microfibrils alternate with lamellae of transverse cellulose microfibrils. This implies that the cyclic reorientation of cortical microtubules (MTs) from longitudinal to transverse and from transverse to longitudinal occurs on the OTW. Treatment with a solution that contained no auxin caused the accumulation of cells with longitudinal MTs, suggesting that auxin is required for the reorientation of MTs from longitudinal to transverse during the reorientation cycle. Treatment with 6-dimethylaminopurine (DMAP), an inhibitor of protein kinases that promoted the reorientation of MTs from transverse to longitudinal, resulted in the accumulation of cells with longitudinal MTs. Subsequent treatment with auxin caused a marked increase in the percentage of cells with transverse MTs and then a decrease in the percentage, indicating that the reorientation of MTs from longitudinal to transverse and then from transverse to longitudinal occurred during treatment with auxin. The percentage of cells with transverse MTs decreased more slowly in segments that had been pretreated with gibberellin A₃ (GA) than in segments that had been pretreated without GA, suggesting that GA, in cooperation with auxin, caused the suppression of the reorientation of MTs from transverse to longitudinal.Abbreviations BL brassinolide - BSA bovine serum albumin - GA gibberellin A₃ - DMAP 6-dimethylaminopurine - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IAA indoleacetic acid - MT microtubule - OTW outer tangential wall - PBS phosphate-buffered saline Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy

Recent studies have indicated that adoptive immunotherapy with autologous antitumor tumor-infiltrating lymphocytes (TILs) following nonmyeloablative chemotherapy mediates tumor regression in approximately 50% of treated patients with metastatic melanoma, and that tumor regression is correlated with the degree of persistence of adoptively transferred T cells in peripheral blood. These findings, which suggested that the proliferative potential of transferred T cells may play a role in clinical responses, led to the current studies in which telomere length as well as phenotypic markers expressed on the administered TILs were examined. TILs that were associated with objective clinical responses following adoptive transfer possessed a mean telomere length of 6.3 kb, whereas TILs that were not associated with significant clinical responses were significantly shorter, averaging 4.9 kb (p < 0.01). Furthermore, individual TIL-derived T cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that were longer than telomeres of T cell clonotypes that failed to persist (6.2 vs 4.5 kb, respectively; p < 0.001). Expression of the costimulatory molecule CD28 also appeared to be associated with long telomeres and T cell persistence. These results, indicating that the telomere length of transferred lymphocytes correlated with in vivo T cell persistence following adoptive transfer, and coupled with the previous observation that T cell persistence was associated with clinical responses in this adoptive immunotherapy trial, suggest that telomere length and the proliferative potential of the transferred T cells may play a significant role in mediating response to adoptive immunotherapy.     

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.     

Physical Exercise Enhances Cognitive Flexibility as Well as Astrocytic and Synaptic Markers in the Medial Prefrontal Cortex

Physical exercise enhances a wide range of cognitive functions in humans. Running-induced cognitive enhancement has also been demonstrated in rodents but with a strong emphasis on tasks that require the hippocampus. Additionally, studies designed to identify mechanisms that underlie cognitive enhancement with physical exercise have focused on running-induced changes in neurons with little attention paid to such changes in astrocytes. To further our understanding of how the brain changes with physical exercise, we investigated whether running alters performance on cognitive tasks that require the prefrontal cortex and whether any such changes are associated with astrocytic, as well as neuronal, plasticity. We found that running enhances performance on cognitive tasks known to rely on the prefrontal cortex. By contrast, we found no such improvement on a cognitive task known to rely on the perirhinal cortex. Moreover, we found that running enhances synaptic, dendritic and astrocytic measures in several brain regions involved in cognition but that changes in the latter measures were more specific to brain regions associated with cognitive improvements. These findings suggest that physical exercise induces widespread plasticity in both neuronal and nonneuronal elements and that both types of changes may be involved in running-induced cognitive enhancement.