bluuebunny/crossref_metadata_2025 huggingface dataset:
Antagonistic control of oxidative stress-induced cell death in
Arabidopsis
by two related, plant-specific zinc finger proteins
Authors: Epple Petra , Mack Amanda A. , Morris Veronica R. F. , Dangl Jeffery L.
Published: May 2003

The most familiar form of plant programmed cell death is the hypersensitive response (HR) associated with successful plant immune responses. HR is preceded by an oxidative burst and the generation of both reactive oxygen intermediates (ROI) and NO. The
Arabidopsis LSD1
gene encodes a negative regulator of plant programmed cell death that meets several criteria for a regulator of processes relevant to ROI management during pathogen responses. Here we demonstrate that a highly conserved
LSD1
paralogue,
LOL1
, acts as a positive regulator of cell death. Manipulation of
LOL1
expression alters both the superoxide-dependent, runaway cell death phenotype of
lsd1
plants and the normal HR. We also show that
LSD1
and
LOL1
have antagonistic effects on copper-zinc superoxide dismutase accumulation, consistent with functions in cell death control via maintenance of ROI homeostasis.
Animal production and farm size in Holmes County, Ohio, and US agriculture
Authors: Bender Martin H.
Published: May 2003

Animal production in US agriculture during 1997 was compared with Holmes County, Ohio, in which half the farms belonged to the agrarian Amish whose small farms have been successful. To compare the intensity of animal production in regard to land that was already devoted solely to domestic feed, the two systems were scaled so that their average farm sizes contained equal land areas devoted to domestic feed and then their animal production per farm was adjusted by the same scaling. By breeding populations, as well as large imports of feed, feeder pigs, calves, and broiler chicks. Holmes County produced three times more milk, four times more broilers, about the same amount of eggs and cattle, and twice the pigs per scaled farm, and hence per given land area, as in the US. Despite the average farm size in Holmes County having been 40% smaller than in Ohio overall, this production yielded more than twice the energy and protein per scaled farm, or per given land area, compared to the US, and required almost twice the feed and 85% as much grazed pasture forage per farm. This was in accord with the fact that feed consumption in Holmes County was twice its harvested crop production, implying a net feed import equal to its crop production. The latter fact was the main contribution to the productivity of Holmes County in excess of the US, and also suggested there would be serious problems in widespread adoption of intensive animal production in regard to agricultural markets, soil fertility and farm nutrient losses through manure application. Energy conversion efficiency for the five animal products and breeding populations was greater in Holmes County than the US (10 and 7%, respectively) and likewise for protein (22 and 13%). Besides imported feed, the higher efficiency of Holmes County was also due to its greater emphasis on milk production, which has benefited from USDA milk price support, modern dairy genetics and dairy nutrition programs. The lower overall efficiency of the US has been partly a result of the fact that beef production and breeding, judged by feed alone, have been the least efficient of the five animal products in energy conversion and nearly the least for protein, regardless of the fact that among the five products, beef cattle are the only animals that nationally derived much of their nutrition from the large national area of grazing land.
Producer satisfaction with returns from farmers' market related activity
Authors: Govindasamy Ramu , Italia John , Zurbriggen Marta , Hossain Ferdaus
Published: May 2003

Over 61% of farmers who sell agricultural products through farmers' markets are satisfied with the returns they generate. Producers who are 50 years of age or older and those who retail at least 70% of the dollar value of the products are more likely to be highly satisfied with their profit margin from farmers' markets. A strong positive relationship is documented between profit margin satisfaction and growers who offer organic produce for sale. Furthermore, the results suggest that producers with farmers' market businesses in the growing stage are more likely to be highly satisfied with profit margins.
Family and seller interactions in farmers' markets in upstate New York
Authors: Baber Laura M. , Frongillo Edward A.
Published: May 2003

Farmers' markets promote alternative agriculture by allowing farmers to meet the demand for specialized agricultural products and establish new businesses. Farmers sell at farmers' markets for social and personal, as well as economic, reasons. Successful farmers' marketing involves interactions with customers, but little is known about these interactions. This study aimed to determine what happens when a family shops at farmers' markets, specifically how parents and children interact in the market in relation to each other and to the farmers. Systematic observation was used to follow 22 families with young children. A coding sheet was used to categorize sensory, perfunctory, informational interactions, attempts to influence purchases and additional interactions. Results showed information was more likely to be exchanged between farmers and parents or parents and children. Children were more likely to be involved in sensory interactions. Farmers interact with families through perfunctory interactions or informational interactions, rather than encouraging them to buy specific items. Since some important societal benefits of farmers' markets can only be achieved if information is exchanged during interactions, market managers and outreach professionals should encourage and assist farmers with providing information to customers. Further research is needed to understand why farmers favor social over explicitly sales-focused interactions, which interactive strategies may be optimal to achieve the fullest economic and non-economic benefits for both farmers and customers, and what is the economic and non-economic impact of providing information in the farmers' market.
Mechanical versus herbicidal strategies for killing a hairy vetch cover crop and controlling weeds in minimum-tillage corn production
Authors: Teasdale John R. , Rosecrance Richard C.
Published: May 2003

The development of production systems that provide the benefits of reduced tillage and also reduce or eliminate herbicide inputs presents a challenge to practitioners of sustainable agriculture. This research was conducted to evaluate management approaches for minimum-tillage corn production in a hairy vetch cover crop, specifically, mechanical versus herbicidal methods for pre-plant cover-crop kill and for post-emergence control of emerged weeds. Pre-plant treatments included 2,4-D [(2,4-dichlorophenoxy)acetic acid] plus residual herbicides, 2,4-D alone, flail mower, corn stalk chopper, light disk and heavy disk. The pre-plant herbicide treatments were followed by a post-emergence treatment of dicamba, whereas the pre-plant mechanical treatments were followed by post-emergence cultivation, as needed, for weed control. The mechanical treatments that kept residue on the soil surface (mower, stalk chopper and light disk) killed hairy vetch when it was flowering, but not when vegetative. The herbicide treatment with pre-plant 2,4-D alone, followed by dicamba post-emergence, controlled annual broadleaf weeds (the dominant species in these experiments) similarly to treatment with 2,4-D plus residual herbicides, suggesting that residual herbicides may be eliminated in cover-crop-based no-tillage systems. The pre-plant mechanical treatments followed by cultivation did not control annual broadleaf weeds as well as herbicide treatments, but did maintain final populations below threshold levels in two of four experiments. The stalk chopper and light disk left high levels of vetch residue on the soil surface, and reduced initial broadleaf weed populations compared with the heavy disk that incorporated residue. However, broadleaf weed populations were reduced with less efficiency by cultivation of unfilled soil following the stalk chopper or light disk (38–69%) than by cultivation of tilled soil following the heavy disk (87–95%). Thus, although maintaining surface cover-crop residue without tillage initially reduced weed emergence, it also reduced the efficiency of cultivation, leading to similar final weed populations in all mechanical-based, pre-plant treatments.
Fructans in the diet cause alterations of intestinal mucosal architecture, released mucins and mucosa-associated bifidobacteria in gnotobiotic rats
Authors: Kleessen Brigitta , Hartmann Ludger , Blaut Michael
Published: May 2003

The effects of fructans in the diet on the mucosal morphometry (height of villi, depth of the crypts, number of goblet cells), the thickness of the epithelial mucus layer and the histochemical composition of intestinal mucosubstances in the distal jejunum and the distal colon were investigated by comparing germ-free (GF) rats, rats harbouringBacteroides vulgatusandBifidobacterium longum(diassociated (DA) rats), and rats with a human faecal flora (HFA). The rats were fed either a commercial standard diet (ST) or ST + (50 g oligofructose (OF)–long-chain inulin (lcIN))/kg. Changes in total bacteria, bifidobacteria andBacteroides–Prevotellain response to feeding these diets were investigated by fluorescentin situhybridization with 16S rRNA-targeted probes both in intestinal contents (lumen bacteria) and tissue sections (mucosa-associated bacteria). The OF–lcIN-containing diet resulted in higher villi and deeper crypts in bacteria-associated, but not in GF rats. In DA and HFA rats, the colonic epithelial mucus layer was thicker and the numbers of the goblet cells were greater than in GF rats. These effects were enhanced by the OF–lcIN-containing diet. In both dietary groups, bacterial colonization of GF rats caused an increase in neutral mucins in the distal jejunum and colon. Bacteria-associated rats had more acidic mucins in the colon than GF rats, and the OF–lcIN-containing diet stimulated sulfomucins as the predominant type of acidic mucins, while sialomucins dominated in the ST-fed groups. The number of mucosa-associated bifidobacteria detected in the colon of DA and HFA rats was greater with OF–lcIN than ST (4·9 and 5·4v. 3·5 and 4·0 log10/mm2mucosal surface respectively), whereas the number of luminal bifidobacteria was only affected by fructans in DA rats.Bacteroidesdid not differ between the groups. The stabilisation of the gut mucosal barrier, either by changes in the mucosal architecture itself, in released mucins or by stimulation of mucosal bifidobacteria with fructans, could become an important topic in the treatment and prophylaxis of gastrointestinal disorders and health maintenance.
Effect of a mixture of conjugated linoleic acid isomers on growth performance and antibody production in broiler chicks
Authors: Takahashi Kazuaki , Akiba Yukio , Iwata Toshio , Kasai Masaaki
Published: May 2003

The effect of dietary conjugated linoleic acid (CLA) isomers mixture on antibody titres against sheep blood erythrocytes (SRBC) and immunoglobulin (Ig) G concentration in plasma was studied in broiler chickens. In experiment 1, male and female broiler chicks (11 d of age, Cobb strain) were fed a diet supplemented with 10 g CLA or 10 g safflower-seed oil/kg diet for 2 weeks. An SRBC suspension (5:100, v/v) in a phosphate buffer was intravenously injected at 18 d of age and a blood sample was taken from the wing vein at 25 d of age. Chicks fed the CLA-supplemented diet had enhanced first antibody titres in plasma to SRBC as compared with those fed the safflower-seed oil-supplemented diet, irrespective of sex differences. In experiment 2, male broiler chicks (8 d of age, Ross strain) were fed a basal diet or a diet containing 10 g CLA/kg diet for 3 weeks. CLA in the CLA diet partially replaced the soyabean oil in the basal diet. The SRBC suspension was intravenously injected at 15 and 25 d of age and a blood sample was obtained at 21 and 29 d of age. The first antibody titres against SRBC were higher in chicks fed the CLA diet than those in chicks fed the basal diet, but the second titres were not. Plasma IgG concentrations in chicks fed the CLA diet were higher than those in chicks fed the basal diet on both sampling days. The results showed that dietary CLA enhanced antibody production in broiler chickens.
Myocardial infarction risk in relation to zinc concentration in toenails
Authors: Martin-Moreno J. M. , Gorgojo L. , Riemersma R. A. , Gomez-Aracen J. , Kark J. D. , Guillen J. , Jimenez J. , Ringstad J. J. , Fernandez-Crehuet J. , Bode P. , Kok F. J. ,   Heavy Metals and Myocardial Infarction Study Group
Published: May 2003

Zn is an essential mineral. The role of Zn in atherosclerosis is not clear. Epidemiological studies, which have reported contradictory results, are limited by the use of serum Zn levels as a marker of intake. We assessed the association of toenail Zn, which integrates dietary Zn intake over 3 to 12 months, with the risk of a first myocardial infarction. Toenail Zn concentrations were determined by neutron activation analysis in the European multi-centre case–control study on antioxidants, myocardial infarction and breast cancer. This multi-centre case–control study included 684 cases and 724 controls from eight European countries and Israel. Toenail Zn levels of controls (adjusted for age and study centre) were positively associated with age, α-tocopherol and Se, but not with additional dietary variables or with classical risk factors for CHD. Average toenail Zn was 106·0 mg/kg in cases (95 % CI 103·1, 108·9) and 107·5 mg/kg in controls (95 % CI 104·5, 110·7). After controlling for cardiovascular risk factors and for centre, the adjusted odds ratios of myocardial infarction for quintiles 2–5 of toenail Zn with respect to the first quintile were 0·97 (95 % CI 0·59, 1·58), 1·15 (95 % CI 0·72, 1·85), 0·91 (95 % CI 0·56, 1·50), and 0·85 (95 % CI 0·52, 1·39). ThePfor trend was 0·45. In conclusion toenail Zn levels (reflecting long-term dietary intake) were not significantly associated with acute myocardial infarction.
Digestion and nutrient net fluxes across the rumen, and the mesenteric- and portal-drained viscera in sheep fed with fresh forage twice daily: net balance and dynamic aspects
Authors: Rémond Didier , Bernard Laurence , Chauveau Béatrice , Nozière Pierre , Poncet Claude
Published: May 2003

Digestion and portal net flux of nutrients were studied in sheep fed twice daily with fresh orchard-grass. Digestive flows were measured in six fistulated sheep using the double-marker technique. Three sheep were fitted with catheters and blood-flow probes, allowing nutrient net flux measurements across the portal-drained viscera (PDV), the mesenteric-drained viscera (MDV) and the rumen. Total tract apparent digestion of N was similar to portal net appearance of N, calculated as the sum of free amino acids (FAA), peptide amino acids (PAA), NH3, and urea net fluxes. PAA accounted for 25 % of non-protein amino acid net release across the PDV. With the exception of glycine and glutamate, the small intestine was the main contributor to this PAA net release. The essential amino acid (EAA) apparent disappearance between the duodenum and the ileum was lower than the net appearance of EAA (FAA + PAA) across the MDV. The value of PDV:MDV flux of free EAA was, on average, 78 %. The rumen accounted for 30 % of the net uptake of EAA by the PDV tissues not drained by the mesenteric vein. Rumen net release of acetate, propionate, butyrate, 3-hydroxybutyrate, and lactate accounted for 70, 55, 46, 77 and 52 %, respectively, of their portal net releases. Conversely, the small intestine was a net consumer of arterial acetate and 3-hydroxybutyrate. Dynamic study of nutrient net fluxes across the PDV showed that throughout a feeding cycle, the liver faced a constant flux of amino acids (AA), whereas volatile fatty acid and NH3net fluxes varied in response to the meal. The present study specified, in forage-fed sheep, the partitioning of nutrient net fluxes across the PDV and the role of peptides in portal net release of AA.
The effect of conjugated linoleic acid and medium-chain fatty acids on transepithelial calcium transport in human intestinal-like Caco-2 cells
Authors: Jewell Christopher , Cashman Kevin D.
Published: May 2003

Capric (10:0) and lauric (12:0) acid and conjugated linoleic acid (CLA) have been shown to increase paracellular permeability across human intestinal-like Caco-2 cell monolayers. While this has generated interest in terms of improved drug absorption and delivery, little has been done in terms of their potential effect on nutrient transport across the intestinal epithelium. Therefore, the objective of the present study was to investigate the effect of these fatty acids on transepithelial Ca transport in Caco-2 cells. Cells were seeded onto permeable transport membranes and allowed to differentiate, over 21 d, into intestinal-like cell monolayers. Monolayers (n9 per treatment) were exposed to 0 (control) or 80 μM-10:0, 80 μM-12:0, 80 μM-18:2, 80 μM-CLA (mixed isomers), 80 μM-cis-9,trans-11 CLA or 80 μM-trans-10,cis-12 CLA for 22 d after seeding (chronic effect) or for 24 h before Ca transport studies (acute effect) on day 22. After exposure, transepithelial and transcellular transport of45Ca, fluorescein transport (a marker of paracellular Ca transport) and transepithelial electrical resistance (TEER, an indicator of permeability) were measured. Overall Ca transport and TEER in Caco-2 cells were unaffected by exposure to any of the fatty acids for 24 h, or to 18:2, CLA blend orcis-9,trans-11 CLA for 22 d. Paracellular (but not total transepithelial and transcellular) Ca transport across Caco-2 cells was significantly increased (P<0·01, by about 1·5-fold relative to the control value) by exposure to 10:0, 12:0 andtrans-10,cis-12 CLA for 22 d, suggesting that these non-esterified fatty acids could potentially enhance Ca absorptionin vivo.
Energy balance in depleted ambulatory patients with chronic obstructive pulmonary disease: the effect of physical activity and oral nutritional supplementation
Authors: Goris Annelies H. C. , Vermeeren Marja A. P. , Wouters Emiel F. M. , Schols Annemie M. W. J. , Westerterp Klaas R.
Published: May 2003

Patients with chronic obstructive pulmonary disease (COPD) often suffer from weight loss. The aim of the present study was to gain insight into the energy balance of depleted ambulatory COPD patients, in relation to their habitual level of physical activity and consumption of oral nutritional supplements. Clinically stable and weight-stable patients (n 20; BMI 19·8± SD 2·0 kg/m2) were studied 1 and 3 months after rehabilitation or recovery in the clinic and were at random assigned to a control or intervention group with regard to nutritional supplementation. Energy intake was measured with a 7 d food record. Energy expenditure was estimated from a simultaneous 7 d assessment of physical activity with a tri-axial accelerometer for movement registration in combination with measured BMR. Body mass was measured at several time points. The body mass remained stable in both groups after 1 or 3 months and mean energy balances were comparable for both groups. The mean body-mass change between month 1 and 3 was negatively related to the mean physical activity level (r −0·49; P=0·03). Weight change over the 3 months was negatively associated with the physical activity level. These results suggest that knowledge about the individual physical activity level is necessary for the estimation of the energy need of the COPD patient.
Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited
Authors: Brown J.K. , Wright S.H. , Miller H.R.P.
Published: May 2003

Mucosal mast cells (MMC) play an important role in the immune response against selected species of intestinal nematode. The kinetics with which different strains of inbred mice resolve infection with Trichinella spiralis correlates with their ability to mount MMC responses in the intestinal mucosa. Homologues of MMC that express and constitutively secrete abundant amounts of the granule chymase, mouse mast cell protease-1 (mMCP-1), can be generated in vitro from bone marrow cultures supplemented with interleukins-3 and -9, stem cell factor and transforming growth factor-β1. Using the enhanced growth characteristics of these MMC homologues, a novel limiting dilution assay for mast cell precursor (MCp) frequency has been developed. The assay is highly specific, in that cultures containing mast cells are identified with mMCP-1 specific antibody, and almost three-fold more sensitive than previously published systems. MCp frequencies were compared in BALB/c and C57/BL10 strains of mice that, respectively, respond rapidly and slowly to infection with T. spiralis. MCp frequency (1/378 bone marrow cells) was significantly greater (P<0.05) in BALB/c than C57/BL10 mice (frequency: 1/751). Similarly the rate of growth of MMC homologues and the production of mMCP-1 was significantly (P<0.05) greater in BALB/c than in C57/BL10 bone marrow cultures.
Serological responses to Ascaris suum adult worm antigens in Iberian finisher pigs
Authors: Frontera E. , Serrano F. , Reina D. , Alcaide M. , Sánchez-López J. , Navarrete I.
Published: May 2003

Adult Ascaris suum were dissected to obtain different worm components (body wall, body fluid, ovaries, uterus and oesophagus) which were used as antigens when testing 95 sera of naturally A. suum-infected Iberian pigs by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). Pigs with patent Ascaris infections had significantly lower ELISA optical density values than pigs without adult worms when using the body fluid and the body wall as antigens. A poor negative correlation was found between adult intestinal worm burden or eggs in faeces and specific antibody responses, measured by ELISA and WB using all antigens. By WB, the recognition of specific bands was variable, but three groups of bands with molecular weights of 97 kDa, 54–58 kDa and 42–44 kDa were generally recognized by sera from naturally infected pigs as well as from hyperimmunized pigs when using the five antigen extracts. The ELISA and WB techniques may be used for immunodiagnosis, using somatic adult worm antigens, to declare young pigs to be Ascaris-free but cannot be used for individual Ascaris-diagnosis in adult Iberian pigs.
Chasing the genes that control resistance to gastrointestinal nematodes
Authors: Behnke J.M. , Iraqi F. , Menge D. , Baker R.L. , Gibson J. , Wakelin D.
Published: May 2003

The host-protective immune response to infection with gastrointestinal (GI) nematodes involves a range of interacting processes that begin with recognition of the parasite's antigens and culminate in an inflammatory reaction in the intestinal mucosa. Precisely which immune effectors are responsible for the loss of specific worms is still not known although many candidate effectors have been proposed. However, it is now clear that many different genes regulate the response and that differences between hosts (fast or strong versus slow or weak responses) can be explained by allelic variation in crucial genes associated with the gene cascade that accompanies the immune response and/or genes encoding constitutively expressed receptor/signalling molecules. Major histocompatibility complex (MHC) genes have been recognized for some time as decisive in controlling immunity, and evidence that non-MHC genes are equally, if not more important in this respect has also been available for two decades. Nevertheless, whilst the former have been mapped in mice, only two candidate loci have been proposed for non-MHC genes and relatively little is known about their roles. Now, with the availability of microsatellite markers, it is possible to exploit linkage mapping techniques to identify quantitative trait loci (QTL) responsible for resistance to GI nematodes. Four QTL for resistance to Heligmosomoides polygyrus, and additional QTL affecting faecal egg production by the worms and the accompanying immune responses, have been identified. Fine mapping and eventually the identification of the genes (and their alleles) underlying QTL for resistance/susceptibility will permit informed searches for homologues in domestic animals, and human beings, through comparative genomic maps. This information in turn will facilitate targeted breeding to improve resistance in domestic animals and, in human beings, focused application of treatment and control strategies for GI nematodes.
Seed ageing of four Western Australian species in relation to storage environment and seed antioxidant activity
Authors: Merritt D.J. , Senaratna T. , Touchell D.H. , Dixon K.W. , Sivasithamparam K.
Published: May 2003

The influence of the storage environment on seed viability and antioxidant potential was examined for four species native to Western Australia: Acacia bivenosa DC., Anigozanthos manglesii D. Don, Banksia ashbyi E.G. Baker, and Mesomelaena tetragona (R. Br.) Benth. Seeds were stored at four water contents (at c. 5%, 11–15%, 20–23% and 50% relative humidity) at each of five temperatures (–196, –18, 5, 23 and 50°C), and seed germination and seedling vigour monitored over an 18-month period. Deterioration was apparent in all species (except A. bivenosa) stored at 50°C, with 11% RH maximizing longevity for B. ashbyi and M. tetragona seeds, and 5% or 11% RH preventing deterioration for A. manglesii seeds. Seed viability generally remained high for all species stored at 23°C or less. Notably, however, germination and seedling vigour of A. manglesii and M. tetragona seeds gradually declined when stored at –18°C, suggesting that storage at this temperature was detrimental. The antioxidant activity of lipid extracts of seeds after 18 months storage at 5, 23 and 50°C was also examined to determine whether the seed viability decline was associated with a loss of antioxidants. Antioxidant activity varied between storage treatments and was not related to seed viability.
Characterization of chitinase activity and gene expression in muskmelon seeds
Authors: Witmer X. , Nonogaki H. , Beers E.P. , Bradford K.J. , Welbaum G.E.
Published: May 2003

Chitinase is often produced in higher plants as a general defence response after wounding or pathogenic attack. Since germinating seeds are exposed to soil pathogens, the activity and expression of chitinase in muskmelon (Cucumis meloL.) seeds was investigated. One acidic and three basic chitinase isoforms were detected, beginning 40 d after anthesis in developing and fully mature seeds. Both acidic and basic chitinase isoforms were found in endosperm tissue during imbibition and after radicle emergence. Basic chitinase isoforms, but not acidic isoforms, were detected in the embryonic axes of imbibed seeds and in seeds before germination, indicating that chitinases are developmentally regulated in specific seed tissues. Two complete cDNAs,Cmchi1andCmchi2, were cloned from germinated muskmelon seeds and are predicted to encode chitinases that show 95% identity to a class III chitinase from cucumber (Cucumis sativusL.) and 61% identity to a class II chitinase from soybean (Glycine maxL.), respectively. Southern blotting indicated thatCmchi2was present only once in the muskmelon genome, whileCmchi1may be present in one or two copies.Cmchi1andCmchi2mRNAs were only detected in radicles of germinating seeds and in roots of mature plants, so additional genes other thanCmchi1andCmchi2must be responsible for the chitinase activity in developing seeds. Salicylic acid and benzothiadiazole stimulated the expression ofCmchi1, but notCmchi2, after radicle emergence. A putative role for chitinase in muskmelon seeds is defence against fungal pathogens.
A novel pathway for MuSK to induce key genes in neuromuscular synapse formation
Authors: Lacazette Eric , Le Calvez Sophie , Gajendran Nadesan , Brenner Hans Rudolf
Published: May 2003

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChRε . We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.
Macrophage podosomes assemble at the leading lamella by growth and fragmentation
Authors: Evans James G. , Correia Ivan , Krasavina Olga , Watson Nicki , Matsudaira Paul
Published: May 2003

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with β-actin–ECFP and L-fimbrin–EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.
The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport
Authors: Bryant Nia J. , James David E.
Published: May 2003

Protein phosphatase 1 (PP1, Glc7p) functions in the final stage of SNARE-mediated vesicle transport between docking and fusion. During this process, trans-SNARE complexes, formed between molecules in opposing membranes, convert to cis-complexes, with all participants in the same lipid bilayer. Here, we show that glc7 mutant cells accumulate SNARE complexes. These complexes are clearly different from those found in either wild-type or sec18–1 cells as the Sec1p/Munc18 (SM) protein Vps45p does not bind to them. Given that PP1 controls fusion, the SNARE complexes that accumulate in glc7 mutants likely represent trans-SNARE complexes. Vps45p dissociates from the membrane in the absence of PP1 activity, but rapidly reassociates after its reactivation. These data reveal that SM proteins cycle on and off membranes in a stage-specific manner during the vesicle transport reaction, and suggest that protein phosphorylation plays a key role in the regulation of this cycle.
Colocalization of synapsin and actin during synaptic vesicle recycling
Authors: Bloom Ona , Evergren Emma , Tomilin Nikolay , Kjaerulff Ole , Löw Peter , Brodin Lennart , Pieribone Vincent A. , Greengard Paul , Shupliakov Oleg
Published: May 2003

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.
Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex
Authors: Nilsson IngMarie , Kelleher Daniel J. , Miao Yiwei , Shao Yuanlong , Kreibich Gert , Gilmore Reid , von Heijne Gunnar , Johnson Arthur E.
Published: May 2003

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61α and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.
Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on theEscherichia coliribosome
Authors: Ullers Ronald S. , Houben Edith N.G. , Raine Amanda , ten Hagen-Jongman Corinne M. , Ehrenberg Måns , Brunner Joseph , Oudega Bauke , Harms Nellie , Luirink Joen
Published: May 2003

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.
The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces
Authors: Bosher Julia M. , Hahn Bum-Soo , Legouis Renaud , Sookhareea Satis , Weimer Robby M. , Gansmuller Anne , Chisholm Andrew D. , Rose Ann M. , Bessereau Jean-Louis , Labouesse Michel
Published: May 2003

Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.
Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin
Authors: Piacentini Lucia , Fanti Laura , Berloco Maria , Perrini Barbara , Pimpinelli Sergio
Published: May 2003

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock–induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock–induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.
Properties of Single Voltage-gated Proton Channels in Human Eosinophils Estimated by Noise Analysis and by Direct Measurement
Authors: Cherny Vladimir V. , Murphy Ricardo , Sokolov Valerij , Levis Richard A. , DeCoursey Thomas E.
Published: May 2003

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi ≤ 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{{\sigma}}_{\mathrm{H}}^{\mathrm{2}}\) \end{document}. We analyze \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{{\sigma}}_{\mathrm{H}}^{\mathrm{2}}\) \end{document} data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi ≤ 5.5 near Vthreshold (the voltage at which gH turns on), single channel–like current events were observed with amplitudes 7–16 fA.
Presenting generalized 𝑞-Schur algebras
Authors: Doty Stephen
Published: May 2003

We obtain a presentation by generators and relations for generalized Schur algebras and their quantizations. This extends earlier results obtained in the type

A
A

case. The presentation is compatible with Lusztig’s modified form

U
˙

\mathbf {\dot {U}}

of a quantized enveloping algebra. We show that generalized Schur algebras inherit a canonical basis from

U
˙

\mathbf {\dot {U}}

, that this gives them a cellular structure, and thus they are quasihereditary over a field.
The Tumor Suppressor cybL, a Component of the Respiratory Chain, Mediates Apoptosis Induction
Authors: Albayrak Timur , Scherhammer Volker , Schoenfeld Nicole , Braziulis Erik , Mund Thomas , Bauer Manuel K.A. , Scheffler Immo E. , Grimm Stefan
Published: May 2003

A genetic screen was established to clone apoptosis-inducing genes in a high-throughput format. It led to the isolation of several proapoptotic genes whose proteins are localized to mitochondria. One of the isolated genes is cytochrome bL(cybL also known as SDHC, CII-3, or QPs-1), a component of the respiratory chain complex II. It was further investigated because both cybL and another component of complex II, cybS, have recently been identified as tumor suppressor proteins, some of which act by controlling apoptosis. Our studies reveal that cell death induction by cybL expression is concomitant with a transient inhibition of complex II and the generation of reactive oxygen species. Importantly, cells that are constitutively deficient in cybL are resistant to a variety of proapoptotic cytostatic drugs and to the effects of the Fas receptor. Our results therefore identify complex II as a sensor for apoptosis induction and could explain the unexpected observation that complex II is inactivated in tumors.
α4β1 Integrin/Ligand Interaction Inhibits α5β1-induced Stress Fibers and Focal Adhesions via Down-Regulation of RhoA and Induces Melanoma Cell Migration
Authors: Moyano José V. , Maqueda Alfredo , Casanova Benito , Garcia-Pardo Angeles
Published: May 2003

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7–10 fragment. Coimmobilization of FNIII4–5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7–10, or addition of soluble FNIII4–5 to cells preattached to FNIII7–10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7–10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7–10+FNIII4–5. Furthermore, addition of alpha4beta1 ligands to FNIII7–10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.
Synergistic Control of Cellular Adhesion by Transmembrane 9 Proteins
Authors: Benghezal Mohammed , Cornillon Sophie , Gebbie Leigh , Alibaud Laeticia , Brückert Franz , Letourneur François , Cosson Pierre
Published: May 2003

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.
Rer1p, a Retrieval Receptor for ER Membrane Proteins, Recognizes Transmembrane Domains in Multiple Modes
Authors: Sato Ken , Sato Miyuki , Nakano Akihiko
Published: May 2003

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Δrer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.