bluuebunny/crossref_metadata_2025 huggingface dataset:
Realizing Low-carbon and Climate-resilient Development on Aquaculture
Authors: Nazar Fitrina , Rahman  , Rifqi Muhammad
Published: February 2024

Aquaculture production supports food security, and it is an important commodity for Indonesia's exports. Climate change determines the sustainability of aquaculture production. Low-carbon development is an effort to control the impact of climate change from a mitigation standpoint, while climate-resilience development is an adaptation. In order to realize sustainable aquaculture in a broad and long dimension and in line with the Sustainable Development Goals (SDGs), it is necessary to develop a low-carbon and climate-resilience development strategy. A descriptive analysis was carried out on a review of published literature to develop the concept of low-carbon and climate-resilience development in aquaculture. The keywords used in the literature search are the impact of climate change on aquaculture, low-carbon development, climate-resilience development, and achievement of the SDGs in aquaculture. The research that has been published mostly discusses the potential Green House Gas emissions (GHG) from aquaculture activities, is still very limited and explains efforts to mitigate and adapt aquaculture to climate change. Efficiency and use of alternative energy sources, increased productivity, feed efficiency and effectiveness, effluent management, disease control and water quality management, and superior seeds can be applied in low-carbon emission development. Meanwhile, climate-resilience development that can be implemented includes the implementation of Integrated Multi-Tropical Aquaculture (IMTA) and Regional Integrated Multi-Tropical Aquaculture (RIMTA), closed systems, and Recirculating Aquaculture Systems (RAS), as well as ponds that integrate mangroves.
Organic wastes of Citrus sinensis Peels- a source of eco-friendly and sustainable bioactive compounds for promoting health
Authors: Olaitan Michael O. , Ujowundu Cosmas O. , Nzebude Chiamaka P. , Ujowundu Favour N. , Ugwu Albert O. , Azuoma Favour C. , Nwokocha George C.
Published: February 2024

To determine the phytochemicals, radical scavenging and antioxidant potential of orange peel extract. Citrus sinensis were subjected to extraction with ethanol. Gas chromatography (GC) was utilized to determine the phytochemical composition of orange peel extract. Hydrogen peroxide, superoxide, nitric oxide, and hydroxyl radical scavenging assays were conducted to assess radical scavenging potential of the extract. Antioxidant activities of the peel extracts were determined via the 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity, ferric reducing antioxidant power (FRAP) assay, ABTS scavenging, and total antioxidant capacity (TAC) assay. The GC-FID analysis revealed the presence of alkaloids, flavonoids, polyphenols, tannins, sapogenin, and steroids in the orange peel extract. The results of radical scavenging assays demonstrated the extract’s ability to scavenge hydrogen peroxide, superoxide, nitric oxide and hydroxyl radicals. The scavenging capacity of the extract was observed to be concentration-dependent, with comparisons made to standard antioxidants ascorbic acid and BHT. Peels from citrus sinensis represent a valuable source of phytochemicals, demonstrating significant antioxidant and radical scavenging activities.
Anthropocene Impacts on Marine Fishers: South Odisha Coast, India
Authors: Mishra Siba Prasad , Mohapatra Saswat
Published: February 2024

This work envisages reporting, detailing, and analyzing the fisher's villages and the fisher's community along the South Odisha coast extending from the Konark (Puri) to Sonapur (Ganjam). The hardworking fisher’s community residing in shanty clusters are economically, politically and socially disadvantaged. They face the challenges of devastating floods, droughts, erosions, high waves, and cyclones consequential in low catch, resulting in migration or marginalization.

The silent combat between orthodox fishing practices and the modern method of synthetic boats/nets has challenged their livelihood, catch, and resources. About 27 coastal villages were surveyed.  The primary data are gathered through questionnaires, interactions and focused group discussions by sampling methods. The secondary latest data is collected from electronic resources, and past literature was analyzed,

The lack of infrastructure, and plastic fishing crafts, banned during mass nesting put the fishers in poverty. The unhealthy living conditions, low economy settings, and non-application of IoT lack of circular economy worsen the setting. Illiteracy, gender parity, nonadaptation of technology and language problems mechanization of fishing art, modernization, societal innovations and the financial assistance for the ban period and adaptation of circular economy, adaptation of sustainability measures can improve their social status.
Design and Construction of a System for Measuring Carbon Monoxide, Hydrogen and Methane Concentrations in a Co-Current Downdraft Biomass Gasifier
Authors: Ousmane Zoundi , Fidele Nzihou Jean , Salou Hamidou , Frederic Ouattara , Gerard Segda Bila
Published: February 2024

Gasification is the process of producing combustible gases from solid materials such as coal, biomass or solid waste. The laboratory of Space Physics and Energy has two experimental gasifiers producing synthetic gases whose natures and concentrations must be determined. To do this, lower-cost sensors were purchased and used for determining the concentration of carbon monoxide with maximum concentration of 1.000 and 2.000 ppm. Hydrogen and methane concentration was not offered by these two commercial gas analyzers. In this paper Metal-Oxide gas sensors were used to extend the measurement range of carbon monoxide up to 4.000ppm.  Hydrogen and methane concentrations up to 10.000ppm in synthetic gas produced by a wood fired co-current downdraft gasifier measurements were also enabled. These sensors have a chemical sensing element based on a layer of tin dioxide (SnO2); whose resistivity is sensitive to nature of the gas between two sensing electrodes. This property gives these sensors a resistive electrical model whose measurand is the concentration of the input gas. This study shows that resistor R of the sensor is related to the gas concentration with an equation of the form: log(R/R0) = A log (x) +B. With A= -0.3072, B = 0.921 for methane, A= -0.6527, B=1.3055 for carbon dioxide and A= -1.522, B = 4.5686 for hydrogen.
Managing and Billing Software for Hypermarket
Authors: N. Baggyalakshmi , N.R. Jokani , R. Revathi
Published: February 2024

It previews the whole system design while in the free demo session and clear your queries by experiencing Optech’s digital dashboard. In the navigation system, we optimize the UI to be straightforward and comprehend for all sets of people so the employees grasp this entire operation. The Optech software has been developed with VB.net and MY SQL Server. The system is efficient in generating reports, which will help in the maintenance of the hypermarket easily. This Optech software can store a lot of records in the database. It’s a user-friendly software, which can be used for service, sales and report. It has an unlimited storage and application can be used in both online and offline. Reports for billing can be generated by data wise or pricewise.
Kabuki syndrome: Clinical data in 20 patients, literature review, and further guidelines for preventive management
Authors: Schrander‐Stumpel Constance Th.R.M. , Spruyt Liesbeth , Curfs Leopold M.G. , Defloor Truus , Schrander Jaap J.P.
Published: December 2004

The Kabuki syndrome, or Niikawa–Kuroki syndrome, is a clinically recognizable syndrome of unknown etiology. Clinical findings include early hypotonia, joint laxity, developmental delay, facial dysmorphism, persistent fetal fingertip pads, cleft palate, hypodontia, lip nodules, heart defects, and a variety of other structural defects. Behavior in general is social and pleasant. In collaboration with the Dutch Kabuki Network, we evaluated the medical data of 20 individuals diagnosed with the syndrome and compared them with data from the literature. In our literature review we used convincing cases only. Frequent findings in the oral region are under‐reported in the literature: apart from the cleft palate (in about 50%), hypodontia with predominantly absence of the upper lateral incisors, and a full lower lip with symmetrical nodules, or (in a minority) lip‐pits are frequent findings. Also under‐reported is the presence of a thickened nuchal fold during pregnancy and hydrops in the neonatal period. Clinical recognition in the neonate is difficult. Towards early puberty acute and serious weight excess has been experienced. We suggest that a cytogenetic abnormality should be ruled out in all cases. We provide further guidelines for preventive management. © 2004 Wiley‐Liss, Inc.
Wild capuchin monkeys (Cebus libidinosus) use anvils and stone pounding tools
Authors: Fragaszy Dorothy , Izar Patrícia , Visalberghi Elisabetta , Ottoni Eduardo B. , de Oliveira Marino Gomes
Published: December 2004

We conducted an exploratory investigation in an area where nut‐cracking by wild capuchin monkeys is common knowledge among local residents. In addition to observing male and female capuchin monkeys using stones to pound open nuts on stone “anvils,” we surveyed the surrounding area and found physical evidence that monkeys cracked nuts on rock outcrops, boulders, and logs (collectively termed anvils). Anvils, which were identified by numerous shallow depressions on the upper surface, the presence of palm shells and debris, and the presence of loose stones of an appropriate size to pound nuts, were present even on the tops of mesas. The stones used to crack nuts can weigh >1 kg, and are remarkably heavy for monkeys that weigh <4 kg. The abundance of shell remains and depressions in the anvil surface at numerous anvil sites indicate that nut‐cracking activity is common and long‐enduring. Many of the stones found on anvils (presumably used to pound nuts) are river pebbles that are not present in the local area we surveyed (except on or near the anvils); therefore, we surmise that they were transported to the anvil sites. Ecologically and behaviorally, nut‐cracking by capuchins appears to have strong parallels to nut‐cracking by wild chimpanzees. The presence of abundant anvil sites, limited alternative food resources, abundance of palms, and the habit of the palms in this region to produce fruit at ground level all likely contribute to the monkeys' routine exploitation of palm nuts via cracking them with stones. This discovery provides a new reference point for discussions regarding the evolution of tool use and material culture in primates. Routine tool use to exploit keystone food resources is not restricted to living great apes and ancestral hominids. Am. J. Primatol. 64:359–366, 2004. © 2004 Wiley‐Liss, Inc.
Use of gesture sequences in chimpanzees
Authors: Liebal Katja , Call Josep , Tomasello Michael
Published: December 2004

Gestural communication in a group of 19 captive chimpanzees (Pan troglodytes) was observed, with particular attention paid to gesture sequences (combinations). A complete inventory of gesture sequences is reported. The majority of these sequences were repetitions of the same gestures, which were often tactile gestures and often occurred in play contexts. Other sequences combined gestures within a modality (visual, auditory, or tactile) or across modalities. The emergence of gesture sequences was ascribed to a recipient's lack of responsiveness rather than a premeditated combination of gestures to increase the efficiency of particular gestures. In terms of audience effects, the chimpanzees were sensitive to the attentional state of the recipient, and therefore used visually‐based gestures mostly when others were already attending, as opposed to tactile gestures, which were used regardless of whether the recipient was attending or not. However, the chimpanzees did not use gesture sequences in which the first gesture served to attract the recipient's visual attention before they produced a second gesture that was visually‐based. Instead, they used other strategies, such as locomoting in front of the recipient, before they produced a visually‐based gesture. Am. J. Primatol. 64:377–396, 2004. © 2004 Wiley‐Liss, Inc.
Sex differences in sexual partner acquisition, retention, and harassment during female homosexual consortships in Japanese macaques
Authors: Vasey Paul L.
Published: December 2004

Female Japanese macaques (Macaca fuscata) in certain populations are unusual in that they exhibit male‐typical patterns of mounting behavior and sexual‐partner preference. The goal of this study was to determine whether female Japanese macaques, from one such population, employ male‐typical behavioral tactics to disrupt existing homosexual consortships, as well as to acquire and retain same‐sex sexual partners. “Harassment” of homosexual consortships occurred when a sexually motivated, third‐party male or female interrupted a consorting female couple by displacing or aggressing them. Sexual harassment was a male‐typical strategy for disrupting existing homosexual consortships, but was rarely performed by females. “Intrusions” occurred when a male or female competitor attempted to acquire exclusive access to a female engaged in a homosexual consortship by targeting that female as the focus of competition and her partner as his/her competitor. “Sexual coercion” occurred when one individual alternately sexually solicited and aggressed another individual as part of the same behavioral sequence during an intrusion. Males employed consortship intrusions and sexual coercion when they attempted to acquire female sexual partners that were already engaged in homosexual consortships, but females rarely did so. However, females did employ male‐typical patterns of aggressive competition and sexual coercion to retain same‐sex sexual partners when confronted with male competitors' attempts to usurp those partners. These results indicate that female sexual activity during homosexual consortships is not uniformly “masculine” in expression, but rather is a mixture of male‐ and female‐typical behaviors. Am. J. Primatol. 64:397–409, 2004. © 2004 Wiley‐Liss, Inc.
Systemic Form of Juvenile Xanthogranuloma: Report of a Case with Liver and Bone Marrow Involvement
Authors: Chantranuwat Chavit
Published: December 2004

Systemic form of juvenile xanthogranuloma with involvement of liver and bone marrow is reported in a 2-month-old female infant who presented with hepatosplenomegaly, severe anemia, and thrombocytopenia. There was no skin lesion, nor bone lesion. The enlarged liver has generalized yellowish spots. The diagnosis of juvenile xanthogranuloma was made by pathologic findings of marrow and portal tract infiltration by S-100 negative, CD1a negative, CD68 positive, and Factor XIIIa positive large pale to foamy histiocytes with Touton giant cells, and lack of Langerhans cell granule by electron microscopic examination. The patient was treated with Vinblastine and Etoposide, and experienced slow and gradual disease regression in one year. To the best of knowledge, this is the first documented case of bone marrow involvement in systemic juvenile xanthogranuloma.
Tumors Associated with Sudden Infant and Childhood Death
Authors: Krous Henry F. , Chadwick Amy E. , Isaacs Hart
Published: December 2004

Tumors are rare causes of sudden death in infancy and early childhood. The goals of this study were to determine the types and frequency of the tumors associated with sudden death occurring in cases between birth and age 3 years. The San Diego Sudden Infant Death Syndrome/sudden unexplained death in childhool (SUDC) Research Project database and the literature were reviewed retrospectively. Sixty-eight cases, with the most (84%) affecting the heart and brain, were identified. Tumors are a rare but significant cause of sudden death in infancy and early childhood, and their diagnosis may have significant genetic implications for planning future pregnancies. The diagnosis of these lesions can be established only after thorough postmortem examination.
Viral Diseases of the Liver in Children: Diagnostic and Differential Diagnostic Considerations
Authors: White Frances V. , Dehner Louis P.
Published: December 2004

This review summarizes the general histologic features of acute and chronic hepatitides and highlights those morphologic findings that may suggest or be diagnostic of a specific agent or etiology. The main epidemiologic, clinical, and pathologic features of the hepatotropic viruses are discussed, with an emphasis on pediatric studies and the differential diagnosis of hepatitis in childhood.
A review of measurements of radionuclides in members of the public in the UK
Authors: Hodgson S A , Ham G J , Youngman M J , Etherington G , Stradling G N
Published: December 2004

This paper summarises a comprehensive review of radio-analytical data from autopsy, whole or partial body monitoring and the assay of teeth, foetuses and urine for non-occupationally exposed members of the public in the UK between 1957 and 2003. Most attention has been given to measurements of artificial radionuclides formed in the nuclear fuel cycle, uranium and thorium.

The review concentrates on measurements on people in the UK who live or have lived in the vicinity of nuclear power sites. When UK data are unavailable, or for the purposes of comparison, information has been included from studies in other countries.

Highlights of key findings of the document are listed:

The concentrations of strontium-90 in bone and teeth have reflected changes in the amounts present in the environment due to fallout from nuclear testing.

There are higher concentration levels of239+240Pu in samples from West Cumbria compared with the rest of the UK. However, the levels are so low that any increase in risk of induced skeletal tumours (including leukaemia) would be very small compared with those arising from the intake of natural radionuclides.

As expected there have been only a few published autopsy studies. Both tissue sample mass and radionuclide concentrations were low, leading to relatively large measurement uncertainties.

Whole body measurements of137Cs in residents in Berkshire and Oxfordshire clearly show the effect of atmospheric testing of nuclear weapons and of the Chernobyl accident.

A survey of whole body137Cs and134Cs content following the Chernobyl accident showed that residents of Central Scotland, North-West England and North Wales had twice the radiocaesium content of residents in the rest of England and Wales.

Measurements of131I in the thyroid have been reported following the accidents at Windscale and Chernobyl for most regions of the UK.

Few excretion studies have been reported although this does not diminish their importance. One study on the urinary excretion rate of90Sr in adults and children living in the Dounreay area suggested that the results did not support this radionuclide as being the cause of increased childhood leukaemia. Similar conclusions were drawn from another study involving the assay of239Pu.

It is suggested that a national database of measurements made on members of the public should be initiated. The database would provide a means for identifying future trends.
Characterization and Expression Analysis of a Serine Acetyltransferase Gene Family Involved in a Key Step of the Sulfur Assimilation Pathway in Arabidopsis
Authors: Kawashima Cintia Goulart , Berkowitz Oliver , Hell Ruediger , Noji Masaaki , Saito Kazuki
Published: December 2004

Ser acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetyl-Ser from l-Ser and acetyl-CoA, leading to synthesis of Cys. According to its position at the decisive junction of the pathways of sulfur assimilation and amino acid metabolism, SATases are subject to regulatory mechanisms to control the flux of Cys synthesis. In Arabidopsis (Arabidopsis thaliana) there are five genes encoding SATase-like proteins. Two isoforms, Serat3;1 and Serat3;2, were characterized with respect to their enzymatic properties, feedback inhibition by l-Cys, and subcellular localization. Functional identity of Serat3;1 and Serat3;2 was established by complementation of a SATase-deficient mutant of Escherichia coli. Cytosolic localization of Serat3;1 and Serat3;2 was confirmed by using fusion construct with the green fluorescent protein. Recombinant Serat3;1 was not inhibited by l-Cys, while Serat3;2 was a strongly feedback-inhibited isoform. Quantification of expression patterns indicated that Serat2;1 is the dominant form expressed in most tissues examined, followed by Serat1;1 and Serat2;2. Although Serat3;1 and Serat3;2 were expressed weakly in most tissues, Serat3;2 expression was significantly induced under sulfur deficiency and cadmium stress as well as during generative developmental stages, implying that Serat3;1 and Serat3;2 have specific roles when plants are subjected to distinct conditions. Transgenic Arabidopsis plants expressing the green fluorescent protein under the control of the five promoters indicated that, in all Serat genes, the expression was predominantly localized in the vascular system, notably in the phloem. These results demonstrate that Arabidopsis employs a complex array of compartment-specific SATase isoforms with distinct enzymatic properties and expression patterns to ensure the provision of Cys in response to developmental and environmental changes.
Molecular Bases for Sensitivity to Acetyl-Coenzyme A Carboxylase Inhibitors in Black-Grass
Authors: Délye Christophe , Zhang Xiao-Qi , Michel Séverine , Matéjicek Annick , Powles Stephen B.
Published: December 2004

In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic acetyl-coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors.
The Role of the Cell Cycle Machinery in Resumption of Postembryonic Development
Authors: Barrôco Rosa Maria , Van Poucke Kris , Bergervoet Jan H.W. , De Veylder Lieven , Groot Steven P.C. , Inzé Dirk , Engler Gilbert
Published: December 2004

Cell cycle activity is required for plant growth and development, but its involvement in the early events that initiate seedling development remains to be clarified. We performed experiments aimed at understanding when cell cycle progression is activated during seed germination, and what its contribution is for proper seedling establishment. To this end, the spatial and temporal expression profiles of a large set of cell cycle control genes in germinating seeds of Arabidopsis (Arabidopsis thaliana) and white cabbage (Brassica oleracea) were analyzed. The in vivo behavior of the microtubular cytoskeleton was monitored during Arabidopsis seed germination. Flow cytometry of Arabidopsis germinating seeds indicated that DNA replication was mainly initiated at the onset of root protrusion, when germination reached its end. Expression analysis of cell cycle genes with mRNA in situ localization, β-glucuronidase assays, and semiquantitative reverse transcription-polymerase chain reaction showed that transcription of most cell cycle genes was detected only after completion of germination. In vivo green fluorescent protein analysis of the microtubule cytoskeleton demonstrated that mitosis-specific microtubule arrays occurred only when the radicle had started to protrude, although the assembly of the microtubular cytoskeleton was promptly activated once germination was initiated. Thus, seed germination involves the synthesis and/or activation of a reduced number of core cell cycle proteins, which only trigger DNA replication, but is not sufficient to drive cells into mitosis. Mitotic divisions are observed only after the radicle has protruded and presumably rely on the de novo production of other cell cycle regulators.
The Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase in Proline Degradation[W]
Authors: Deuschle Karen , Funck Dietmar , Forlani Giuseppe , Stransky Harald , Biehl Alexander , Leister Dario , van der Graaff Eric , Kunze Reinhard , Frommer Wolf B.
Published: December 2004

In response to stress, plants accumulate Pro, requiring degradation after release from adverse conditions. Δ1-Pyrroline-5-carboxylate dehydrogenase (P5CDH), the second enzyme for Pro degradation, is encoded by a single gene expressed ubiquitously. To study the physiological function of P5CDH, T-DNA insertion mutants in AtP5CDH were isolated and characterized. Although Pro degradation was undetectable in p5cdh mutants, neither increased Pro levels nor an altered growth phenotype were observed under normal conditions. Thus AtP5CDH is essential for Pro degradation but not required for vegetative plant growth. External Pro application caused programmed cell death, with callose deposition, reactive oxygen species production, and DNA laddering, involving a salicylic acid signal transduction pathway. p5cdh mutants were hypersensitive toward Pro and other molecules producing P5C, such as Arg and Orn. Pro levels were the same in the wild type and mutants, but P5C was detectable only in p5cdh mutants, indicating that P5C accumulation may be the cause for Pro hypersensitivity. Accordingly, overexpression of AtP5CDH resulted in decreased sensitivity to externally supplied Pro. Thus, Pro and P5C/Glu semialdehyde may serve as a link between stress responses and cell death.
Encounters between Dynamic Cortical Microtubules Promote Ordering of the Cortical Array through Angle-Dependent Modifications of Microtubule Behavior[W]
Authors: Dixit Ram , Cyr Richard
Published: December 2004

Ordered cortical microtubule arrays are essential for normal plant morphogenesis, but how these arrays form is unclear. The dynamics of individual cortical microtubules are stochastic and cannot fully account for the observed order; however, using tobacco (Nicotiana tabacum) cells expressing either the MBD-DsRed (microtubule binding domain of the mammalian MAP4 fused to the Discosoma sp red fluorescent protein) or YFP-TUA6 (yellow fluorescent protein fused to the Arabidopsis α-tubulin 6 isoform) microtubule markers, we identified intermicrotubule interactions that modify their stochastic behaviors. The intermicrotubule interactions occur when the growing plus-ends of cortical microtubules encounter previously existing cortical microtubules. Importantly, the outcome of such encounters depends on the angle at which they occur: steep-angle collisions are characterized by approximately sevenfold shorter microtubule contact times compared with shallow-angle encounters, and steep-angle collisions are twice as likely to result in microtubule depolymerization. Hence, steep-angle collisions promote microtubule destabilization, whereas shallow-angle encounters promote both microtubule stabilization and coalignment. Monte Carlo modeling of the behavior of simulated microtubules, according to the observed behavior of transverse and longitudinally oriented cortical microtubules in cells, reveals that these simple rules for intermicrotubule interactions are necessary and sufficient to facilitate the self-organization of dynamic microtubules into a parallel configuration.
The Vegetative Vacuole Proteome ofArabidopsis thalianaReveals Predicted and Unexpected Proteins[W]
Authors: Carter Clay , Pan Songqin , Zouhar Jan , Avila Emily L. , Girke Thomas , Raikhel Natasha V.
Published: December 2004

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.
Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells
Authors: Ikegami Machiko , Na Cheng-Lun , Korfhagen Thomas R. , Whitsett Jeffrey A.
Published: December 2004

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(−/−)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(−/−) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(−/−), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(−/−) mice. SA from SP-D(−/−) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(−/−) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(−/−) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.
Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation
Authors: Prado Carla M. , Leick-Maldonado Edna A. , Arata Vanessa , Kasahara David I. , Martins Mílton A. , Tibério Iolanda F. L. C.
Published: December 2004

In the present study we evaluated the role of neurokinins in the modulation of inducible nitric oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this response. Animals were anesthetized and pretreated with capsaicin (50 mg/kg sc) or vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for 4 wk. Animals were then anesthetized, mechanically ventilated, given normal saline or NG-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg ic), and challenged with ovalbumin. Prechallenge exhaled NO increased in ovalbumin-exposed guinea pigs ( P < 0.05 compared with controls), and capsaicin reduced this response ( P < 0.001). Compared with animals inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory system resistance and elastance and numbers of total mononuclear cells and eosinophils, including those expressing iNOS ( P < 0.001). Capsaicin reduced all these responses ( P < 0.05) except for iNOS expression in eosinophils. Treatment with l-NAME increased postantigen challenge elastance and restored both resistance and elastance previously attenuated by capsaicin treatment. Isolated l-NAME administration also reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS ( P < 0.05 compared with ovalbumin alone). Because l-NAME treatment restored lung mechanical alterations previously attenuated by capsaicin, NO and neurokinins may interact in controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil and lymphocyte infiltration in the airways.
Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema
Authors: Plantier Laurent , Marchand-Adam Sylvain , Marchal-Sommé Joëlle , Lesèche Guy , Fournier Michel , Dehoux Monique , Aubier Michel , Crestani Bruno
Published: December 2004

Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.52 pg/μg, range of 0.15–7.40 pg/μg) than in control fibroblasts (14.16 pg/μg, range of 2.50–47.62 pg/μg; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/μg protein, range of 10.4–39.2 pg/μg) was lower than that of controls at baseline (57.5 pg/μg, range of 20.4–116 pg/μg; P = 0.019) and after stimulation with interleukin-1β or prostaglandin E2. Neither retinoic acids (all- trans and 9- cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/μg, range of 2.2–9.3 pg/μg) was similar to that of controls at baseline (2.6 pg/μg, range of 1–6.1 pg/μg; P = 0.14) but could not be stimulated with interleukin-1β. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.
The murine SP-C promoter directs type II cell-specific expression in transgenic mice
Authors: Glasser Stephan W. , Eszterhas Susan K. , Detmer Emily A. , Maxfield Melissa D. , Korfhagen Thomas R.
Published: December 2004

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.
In vivo inosine protects alveolar epithelial type 2 cells against hyperoxia-induced DNA damage through MAP kinase signaling
Authors: Buckley S. , Barsky L. , Weinberg K. , Warburton D.
Published: December 2004

Inosine, a naturally occurring purine with anti-inflammatory properties, was assessed as a possible modulator of hyperoxic damage to the pulmonary alveolar epithelium. Rats were treated with inosine, 200 mg/kg ip, twice daily during 48-h exposure to >90% oxygen. The alveolar epithelial type 2 cells (AEC2) were then isolated and cultured. AEC2 isolated from inosine-treated hyperoxic rats had less DNA damage and had increased antioxidant status compared with AEC2 from hyperoxic rats. Inosine treatment during hyperoxia also reduced the proportion of AEC2 in S and G2/M phases of the cell cycle and increased levels of the DNA repair enzyme 8-oxoguanine DNA glycosylase. Bronchoalveolar lavage (BAL) recovered from hyperoxic, inosine-treated rats contained threefold higher levels of active transforming growth factor-β than BAL from rats exposed to hyperoxia alone, and Smad2 was activated in AEC2 isolated from these animals. ERK1/2 was activated both in freshly isolated and 24-h-cultured AEC2 by in vivo inosine treatment, whereas blockade of the MAPK pathway in vitro reduced the protective effect of in the vivo inosine treatment. Together, the data suggest that inosine treatment during hyperoxic exposure results in protective signaling mediated through pathways downstream of MEK. Thus inosine may deserve further evaluation for its potential to reduce hyperoxic damage to the pulmonary alveolar epithelium.
Pulmonary hypertension impairs alveolarization and reduces lung growth in the ovine fetus
Authors: Grover Theresa R. , Parker Thomas A. , Balasubramaniam Vivek , Markham Neil E. , Abman Steven H.
Published: December 2004

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical disorder characterized by abnormal vascular structure, growth, and reactivity. Disruption of vascular growth during early postnatal lung development impairs alveolarization, and newborns with lung hypoplasia often have severe pulmonary hypertension. To determine whether pulmonary hypertension can directly impair vascular growth and alveolarization in the fetus, we studied the effects of chronic intrauterine pulmonary hypertension on lung growth in fetal lambs. We performed surgery, which included partial constriction of the ductus arteriosus (DA) to induce pulmonary hypertension (PH, n = 14) or sham surgery (controls, n = 13) in fetal lambs at 112–125 days (term = 147 days). Tissues were harvested near term for measurement of right ventricular hypertrophy (RVH), radial alveolar counts (RAC), mean linear intercepts (MLI), wall thickness, and vessel density of small pulmonary arteries. Chronic DA constriction caused RVH ( P < 0.0001), increased wall thickness of small pulmonary arteries ( P < 0.002), and reduced small pulmonary artery density ( P < 0.005). PH also reduced alveolarization, causing a 27% reduction in RAC and 20% increase in MLI. Furthermore, prolonged DA constriction (21 days) not only decreased RAC and increased MLI by 30% but also caused a 25% reduction of lung-body weight ratio. We conclude that chronic PH reduces pulmonary arterial growth, decreases alveolar complexity, and impairs lung growth. We speculate that chronic hypertension impairs vascular growth, which disrupts critical signaling pathways regulating lung vascular and alveolar development, thereby interfering with alveolarization and ultimately resulting in lung hypoplasia.
Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia
Authors: Koo Hshi-chi , Davis Jonathan M. , Li Yuchi , Hatzis Dimitrios , Opsimos Harry , Pollack Simcha , Strayer Marlene S. , Ballard Philip L. , Kazzaz Jeffrey A.
Published: December 2004

Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50–300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O2 for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells ( P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD ( P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.
Difference in aftereffects following prolonged Achilles tendon vibration on muscle activity during maximal voluntary contraction among plantar flexor synergists
Authors: Ushiyama Junichi , Masani Kei , Kouzaki Motoki , Kanehisa Hiroaki , Fukunaga Tetsuo
Published: December 2004

It has been suggested that a suppression of maximal voluntary contraction (MVC) induced by prolonged vibration is due to an attenuation of Ia afferent activity. The purpose of the present study was to test the hypothesis that aftereffects following prolonged vibration on muscle activity during MVC differ among plantar flexor synergists owing to a supposed difference in muscle fiber composition. The plantar flexion MVC torque and surface electromyogram (EMG) of the medial head of gastrocnemius (MG), the lateral head of gastrocnemius (LG), and the soleus (Sol) were recorded in 13 subjects before and after prolonged vibration applied to the Achilles tendon at 100 Hz for 30 min. The maximal H reflexes and M waves were also determined from the three muscles, and the ratio between H reflexes and M waves (H/Mmax) was calculated before and after the vibration. The MVC torque was decreased by 16.6 ± 3.7% after the vibration ( P < 0.05; ANOVA). The H/Mmax also decreased for all three muscles, indicating that Ia afferent activity was successfully attenuated by the vibration in all plantar flexors. However, a reduction of EMG during MVC was observed only in MG (12.7 ± 4.0%) and LG (11.4 ± 3.9%) ( P < 0.05; ANOVA), not in Sol (3.4 ± 3.0%). These results demonstrated that prolonged vibration-induced MVC suppression was attributable mainly to the reduction of muscle activity in MG and LG, both of which have a larger proportion of fast-twitch muscle fibers than Sol. This finding suggests that Ia-afferent activity that reinforces the recruitment of high-threshold motor units is necessary to enhance force exertion during MVC.
Effect of phosphorylation by AMP-activated protein kinase on palmitoyl-CoA inhibition of skeletal muscle acetyl-CoA carboxylase
Authors: Rubink D. S. , Winder W. W.
Published: December 2004

AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and inactivate skeletal muscle acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 and fatty acid oxidation. Contraction-induced activation of AMPK with subsequent phosphorylation/inactivation of ACC has been postulated to be responsible in part for the increase in fatty acid oxidation that occurs in muscle during exercise. These studies were designed to answer the question: Does phosphorylation of ACC by AMPK make palmitoyl-CoA a more effective inhibitor of ACC? Purified rat muscle ACC was subjected to phosphorylation by AMPK. Activity was determined on nonphosphorylated and phosphorylated ACC preparations at acetyl-CoA concentrations ranging from 2 to 500 μM and at palmitoyl-CoA concentrations ranging from 0 to 100 μM. Phosphorylation resulted in a significant decline in the substrate saturation curve at all palmitoyl-CoA concentrations. The inhibitor constant for palmitoyl-CoA inhibition of ACC was reduced from 1.7 ± 0.25 to 0.85 ± 0.13 μM as a consequence of phosphorylation. At 0.5 mM citrate, ACC activity was reduced to 13% of control values in response to the combination of phosphorylation and 10 μM palmitoyl-CoA. Skeletal muscle ACC is more potently inhibited by palmitoyl-CoA after having been phosphorylated by AMPK. This may contribute to low-muscle malonyl-CoA values and increasing fatty acid oxidation rates during long-term exercise when plasma fatty acid concentrations are elevated.
Occlusion of the upper airway does not augment the cardiovascular response to arousal from sleep in humans
Authors: O'Driscoll Denise M. , Kostikas Konstantinos , Simonds Anita K. , Morrell Mary J.
Published: December 2004

The cardiovascular response to an arousal from sleep at the termination of an obstructive apnea is more than double that to a spontaneous arousal. We investigated the hypothesis that stimulation of respiratory mechanoreceptors, by inspiring against an occluded airway during an arousal from sleep, augments the accompanying cardiovascular response. Arousals (>10 s) from stage 2 sleep were induced by a 1-s auditory tone (85 dB) during a concomitant 1-s inspiratory occlusion (O) and without an occlusion [i.e., control arousal (C)] in 15 healthy men (mean ± SE: age, 25 ± 1 yr). Arousals were associated with a significant increase in mean arterial blood pressure (MAP) at 4 s ( P < 0.001) and a significant decrease in R-R interval at 3 s ( P < 0.001). However, the magnitude of the cardiovascular response was not different during C compared with O (MAP: C, 86 ± 3 to 104 ± 3 mmHg; O, 86 ± 3 to 105 ± 3 mmHg; P = 0.99. R-R interval: C, 1.12 ± 0.03 to 0.89 ± 0.04 s; O, 1.11 ± 0.02 to 0.87 ± 0.02 s, P = 0.99). Ventilation significantly increased during arousals under both conditions at the second breath ( P < 0.001); this increase was not different between the two conditions (C: 4.40 ± 0.29 to 6.76 ± 0.61 l/min, O: 4.35 ± 0.34 to 7.65 ± 0.73 l/min; P = 0.31). We conclude that stimulation of the respiratory mechanoreceptors by transient upper airway occlusion is unlikely to interact with the arousal-related autonomic outflow to augment the cardiovascular response in healthy young men.