bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual transduction of insulin‐like growth factor‐I and interleukin‐l receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model
Authors: Haupt Jennifer L. , Frisbie David D. , McIlwraith C. Wayne , Robbins Paul D. , Ghivizzani Steve , Evans Chris H. , Nixon Alan J.
Published: September 2004

This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin‐like growth factor‐I (AdIGF‐I) and interleukin‐1 receptor antagonist protein (AdIL‐1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF‐I at 200 MOI in 500 μl serum‐free medium; (3) transduced with AdIL‐1Ra at 100 MOI; or (4) transduced with a combination of AdIGF‐I (200 MOI) and AdIL‐1Ra (100 MOI). Following transduction, cartilage explants were exposed to the synovial monolayer medium using co‐culture inserts. Cultures were maintained for 6 days in either serum‐free medium or medium containing 10ng/ml recombinant human interleukin‐1β. At termination, synovial cell RNA was isolated for real‐time PCR analysis, and cartilage explants were collected for H&E and toluidine blue staining, immunohistochemistry for type II collagen and IGF‐I, in situ localization of IGF‐I and type II collagen gene expression, and biochemical assays. Synovial monolayers were readily transduced with both AdIGF‐I and AdIL‐1Ra. IGF‐I and IL‐1Ra protein were secreted at beneficial levels throughout the experiment, having peak concentrations of 94.6ng/ml and 33.0ng/ml, respectively. Transduction with IGF‐I promoted cartilage production of proteoglycan and type II collagen, suggesting a beneficial role for healing injured cartilage. Transduction with IL‐1Ra decreased the synovial expression of IL‐1α and IL‐1β and matrix metalloproteinases, indicating a mechanism for prevention of matrix degradation. The beneficial effects of the combination of anabolic growth factors and catabolic blockers were evident in improved preservation of proteoglycan content of cartilage explants exposed to the depleting effects of IL‐1. These results show that gene therapy combining anabolic growth factors to stimulate matrix synthesis and catabolic blockers to prevent matrix degradation by IL‐1, protects and causes partial restoration of cartilage matrix, and suggest a potential benefit of combination gene therapy for cartilage healing. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Latin and English as world languages
Authors: Wright Roger
Published: September 2004

Can we learn from what happened to Latin, in terms of its native speakers and foreign users? Comparisons are often made between the role of Latin during and after the Roman Empire and the role of English in the present. These can often be illuminating, particularly for the student of the sociolinguistics of the Late Latin-speaking world, where a generous application of the uniformitarian principle allows us to avoid now some of the misunderstandings that were common in the past: for example, the realization that linguistic change is inevitable and in itself neither good nor bad, and that language-internal variation is not pathological, and need not necessarily in itself lead to fragmentation, has been salutary. As a result, the modern view of the development of Latin into Romance, and of Romance into the separate Romance languages, is almost certainly more plausible now than it used to be. We have a more nuanced account to present, even though there is a great deal we do not and perhaps cannot know, including in particular an inability to be sure about the dating of developments which we can be sure occurred at some point.
Persian loanwords in English
Authors: Kaye Alan S.
Published: September 2004

A survey of terms inherited from Persian, often through intermediate languages. ENGLISH may be considered a typical case of a European language indebted to Persian, often through the intermediary of another language, a point with which this survey seeks to deal. A comprehensive list of such loanwords, direct and indirect, can be found in Cannon and Kaye (2001), to which the reader is referred for the full list of the 811 items, including their etyma. One hundred and thirty three of these are distant loans, as with the word azure (‘a light purplish blue’), which comes into English through Old French, probably through Old Spanish azur ∼ azul, which comes in turn from Arabic lazaward ∼ lazuward, and ultimately from Persian lajuvard (‘azure; cobalt blue’). This thematically-organized article provides a general review of the field.
So what's in a book?
Authors: Blaisdell Bob
Published: September 2004

Three linguistical tales from New York.
‘Is English we speaking’: Trinbagonian in the twenty-first century
Authors: Youssef Valerie
Published: September 2004

Some notes and comments on the English usage of Trinidad and Tobago. The paper argues that for Trinidadians to think in terms of speaking and owning ‘only’ their distinctive Creole, setting aside their long-established indigenous variety of Standard English as if not really their own, is a complex distortion of social and linguistic reality. The reality has emerged from social, cultural, and psychological factors present in the Anglophone Caribbean at large, includes both conventional English and the Creole with which it inter-operates on a daily basis, and is an issue that stands in need of a positive revision that acknowledges the islands' dual inheritance.
Consonantal beginnings
Authors: Bulley Michael
Published: September 2004

The writer discusses possible pairings of consonants at the beginnings of English words. (A revision of the article that first appeared in ET79.)
Spotted hyenas (Crocuta crocuta) follow migratory prey. Seasonal expansion of a clan territory in Etosha, Namibia
Authors: Trinkel Martina , Fleischmann Paul H. , Steindorfer Albert F. , Kastberger Gerald
Published: September 2004

The spatial organization of one clan of spotted hyenas Crocuta crocuta in the centre of the Etosha National Park, Namibia, is described during the dry and the wet seasons. The clan comprised 11 adults and sub‐adults and occupied a territory of 160 km2 in the dry season and 320 km2 in the wet season. The dry season territory contained a low density (one animal/km2) of resident herbivores, such as gemsbok Oryx gazella, kudu Tragelaphus strepsiceros, giraffe Giraffa camelopardis, steenbok Raphicerus campestris and ostrich Struthio camelus, and a higher density of migratory species (12 animals/km2), principally springbok Antidorcas marsupialis, zebra Equus burchelli and wildebeest Connochaetes taurinus. These migratory species were the main prey of clan members. At the start of the wet season, the migratory herbivores migrated to the north‐west, resulting in a considerable decline in the density of prey in the area used by clan members during the dry season. In response to this decline in prey, clan members followed the migratory herds and shifted the focus of their activities to an area grazed by migratory herbivores during the wet season. There existed a strong spatial relationship between the hyena density and the migratory prey density in both the dry and wet season. We postulate that the considerable enlargement of the clan territory in the wet season is a response to both the migratory movements of prey and an increase in the dispersion of prey during the wet season.
Patterns of reproductive effort in male ungulates
Authors: Mysterud Atle , Langvatn Rolf , Stenseth Nils Chr.
Published: September 2004

In ungulates, males and females have contrasting life histories, as usually only the females raise the young. How reproductive effort in males varies with individual level and population level characteristics has received little attention in the literature. Using published information on direct (weight loss during the rut) and indirect (rut‐related changes in activity budgets, fighting frequency, etc.) measures of reproductive effort, we tested whether effort in males increased with (H1) increasing age, (H2) increasing body size, (H3) decreasing population density, (H4) increasingly female‐biased sex ratio and with a younger male age structure. Consistent with H1, reproductive effort was consistently higher in prime‐aged than in younger males in a large number of studies. Among younger males, sub‐adult males had an equal or higher effort than yearling males. Prime‐aged males had more typical rutting behaviour (e.g. roaring, tending, fighting and chasing frequencies) and they often lowered their intake of forage during the rut. However, reproductive effort usually declined for very old age classes. Reproductive effort increased with size (H2) also after accounting for age. Data on effort vs (H3) density and (H4) sex ratio/male age structure were inconclusive. Clearly, far more studies relating effort to population characteristics are needed before the rutting ecology of male ungulates can be understood.
Heritable variation in testosterone levels in male garter snakes (Thamnophis sirtalis)
Authors: King R. B. , Cline J. H. , Hubbard C. J.
Published: September 2004

Circulating testosterone levels were measured at 195, 318 and 437 days of age in 76 male garter snakes (Thamnophis sirtalis) born to 26 wild‐caught females. Testosterone levels increased significantly with increasing mass at all three ages and differed significantly among families at 318 and 437 days.‘Full‐sib’ estimates of heritability were near one at 318 and 437 days but these estimates may be inflated by maternal effects, dominance and epistasis. Evidence of heritable variation in circulating testosterone levels suggests that the evolution of traits affected by this hormone (reproductive, territorial and agonistic behaviour; body size and shape) might be mediated through changes in this hormonal control mechanism.
A new species of bandicoot,Microperoryctes aplini, from western New Guinea
Authors: Helgen Kristofer M. , Flannery Timothy F.
Published: September 2004

A distinctive new striped bandicoot of the genusMicroperoryctes (M. aplininew species) is described and compared withMicroperoryctes murina, another little‐known bandicoot with which the new species has been confused in the past. The smallest of all living bandicoots,M. apliniis apparently restricted to high‐elevation forests in the Arfak Mountains of the Vogelkop Peninsula of western New Guinea. The similarly smallM. murinais known only from high‐elevation forests on Gunung Sumuri, a peak in the Weyland Range of western New Guinea. The two species differ markedly in fur colour and ornamentation, pelage texture, external proportions, and quantitative and qualitative craniodental aspects, and may not be immediately related to one another.
The chronology and frequency of a stress marker (linear enamel hypoplasia) in recent and archaeological populations ofSus scrofain north‐west Europe, and the effects of early domestication
Authors: Dobney Keith , Ervynck Anton , Albarella Umberto , Rowley‐Conwy Peter
Published: September 2004

Linear enamel hypoplasia (LEH), a deficiency in enamel formation visible on mammal tooth crowns, can be used as a retrospective indicator of physiological stress and developmental health in humans and animals. In this first study, for north‐western Europe, the condition has been recorded from prehistoric (mesolithic) and recent populations of wild boar, and from domestic pigs belonging to early farming (neolithic) communities. It was possible to show that LEH occurs in recent and ancient populations of wild boar from north‐west Europe, and that the occurrence of the condition can be explained by the same events within the animal's life (birth, weaning, winter starvation) as has been previously suggested for archaeological domestic pig samples. The frequency of LEH is consistently low within all ancient and recent populations of wild boar studied, a remarkable observation given the pronounced differences in the living conditions of these two diachronically well‐separated groups, mainly linked with the increasing human pressure on recent populations of wild animals. Early domestic samples generally show high LEH frequencies, although considerable variation exists between the samples. It is suggested that these high frequencies are, in general, the result of domestication, while the variation could be related to differences in early husbandry. The observation of LEH, therefore, provides a valuable tool for studying the history of animal domestication.
Survey of the gastrointestinal parasites of the primate community at Tambopata National Reserve, Peru
Authors: Phillips Kimberley A. , Haas Meghan E. , Grafton Brian W. , Yrivarren Mirtha
Published: September 2004

The main objective of this project was to provide baseline data on faecal parasites of groups of non‐human primates from Tambopata Research Center, Tambopata National Reserve, Peru. All primate species found in this area were sampled: red howler monkeys Alouatta seniculus, night monkeys Aotus vociferans, spider monkeys Ateles bezlebuth chamek, brown titi monkeys Callicebus brunneus, white‐fronted capuchins Cebus albifrons, brown capuchins Cebus apella, saddleback tamarins Saguinus fuscicollis and squirrel monkeys Saimiri sciureus. Individuals from four howler monkey troops, three brown titi monkey troops, two squirrel monkey troops and one troop each of night monkeys, spider monkeys, brown capuchins, white‐fronted capuchins and saddleback tamarins were sampled. Faecal samples were collected from July to October 2002 from 86 individuals. A concentration test was used to analyse faecal samples. Results indicate the presence of various protozoans, Ancyclostoma sp., Ascaris sp., Strongyloides stercoralis, Trichuris trichiura, Prosthenorchis elegans and Schistosoma mansoni.
Selective consumption by predators of different body regions of prey: is rate of energy intake important?
Authors: Chen Xiaolin , Dickman Chris R. , Thompson Michael B.
Published: September 2004

If the energy‐density of prey bodies is not uniform, predators usually should maximize their rate of energy intake while foraging by selectively consuming energy‐rich regions of their prey and discarding other parts. In this study, the hypothesis of selective body‐part consumption was tested using two species of dasyurid marsupials, Sminthopsis youngsoni and Ningaui ridei, and their invertebrate prey in arid central Australia. Energy‐densities were similar for several divergent types of prey, including whole insects, centipedes, and spiders (20.2 ± 2.19 (sd) J/mg ash‐free dry mass), but there were marked differences in energy‐density among orders of insects and also between different body regions of invertebrates. In captivity, the two dasyurid species consumed different body regions of prey in the same way. For beetles and cockroaches, the rank order for selection of body parts was abdomen, thorax, head, then legs; for centipedes it was body, then head + legs; and for spiders it was opisthosoma, prosoma and then legs. Selection of prey body regions by the marsupials correlated closely with the energy‐densities of these regions, and also with the rates of energy intake that they yielded. In the field, selection of body parts of arthropod prey by dasyurids probably occurs primarily when prey is abundant. This selection should allow maximization of rates of energy intake in favourable periods and should, in turn, allow dasyurid predators to effectively exploit pulses of prey resources in the temporally variable desert environment.
Digestive strategies in two sympatrically occurring lagomorphs
Authors: Kuijper D. P. J. , van Wieren S. E. , Bakker J. P.
Published: September 2004

Separation of low digestible fibres and fermentation of the digestible part of the food in the caecum is an adaptation of some small herbivores to cope with low‐quality forage. The caecum content is later re‐ingested as soft faeces so that the herbivore can benefit from this protein‐rich material. This is known as caecotrophy and is a common phenomenon in species of leporids, although differences exist between hares and rabbits. Hares have amorphous soft faeces and the amount of soft faeces produced is smaller compared to that of rabbits. Both factors suggest that hares have smaller benefits from re‐ingestion of the caecal contents compared with rabbits and, as a consequence, have a less efficient digestion (mainly of nitrogen) compared to rabbits. The assertion was tested whether digestive efficiency is different between the two herbivores and how this affects the choice of food plants in a natural situation. A feeding trial was conducted using hares and rabbits fed with diets with a range of fibre contents. Dry matter digestibility was not different, but nitrogen digestibility was lower in hares than in rabbits, indicating a less efficient digestion of protein. Both taxa showed a different response to increased fibre content in the diet. Rabbits maximized digestibility by increasing retention time of the food, hares maximized digestion rate by increasing the passage rate of the food through the digestive tract. The daily digestible nitrogen intake was higher in hares Lepus europaeus than that in rabbits Oryctolagus cuniculus, indicating that hares compensated for their lower nitrogen digestibility. Hares were predicted to select for higher quality plant species in a natural situation, but they had, on average, a lower nitrogen and higher total fibre content in their diet compared to sympatrically occurring rabbits. This indicated that hares did not compensate for their lower digestive efficiency by selecting higher quality food plants. The present experiment shows that hares and rabbits have different digestive strategies to cope with low quality forage. Rabbits had a higher N‐digestibility by increasing the retention time, whereas hares appeared to compensate for their lower N‐digestibility by increasing the processing rate, when food quality deteriorated.
Litigation and locality: the Cambridge university courts, 1560–1640
Authors: SHEPARD ALEXANDRA
Published: September 2004

The importance of legal institutions as mediators of social relations in early modern towns has long been recognized. However, opinion differs over the extent to which early modern courts generated social conflict or resolved it through promoting consensus. This article brings to light a neglected jurisdiction and argues that while the university courts inevitably generated conflict when pursuing their regulative agenda, they nonetheless offered Cambridge inhabitants a considerable resource which was used extensively in both the speedy resolution and the vexatious prolongation of a wide range of disputes which tended to cut across rather than deepen town–gown hostilities.
Satellite observations of spatial and interannual variability of lightning and radar reflectivity
Authors: Durden S. L. , Meagher J. P. , Haddad Z. S.
Published: September 2004

The authors use satellite data to examine the relationship between lightning and upper‐level radar reflectivity. They find correlations between average flash rates and upper‐level reflectivities over both land and ocean, although both flash rates and reflectivities are much lower over ocean than land. Analysis of the data using Empirical Orthogonal Functions (EOFs) shows similar EOFs for averaged lightning and reflectivity. In contrast, the EOFs of the anomalies of lightning and reflectivity have different spatial patterns; however, both have principal component time series that are correlated with the Southern Oscillation Index and, hence, El Niño. Differences in behavior of the lightning and reflectivity anomaly EOFs and principal components suggest that El Niño plays a smaller role in lightning anomaly than precipitation anomaly.
Decadal weakening of the shallow overturning circulation in the South Indian Ocean
Authors: Lee Tong
Published: September 2004

Satellite observations reveal a substantial weakening of the southeasterly trade wind over the South Indian Ocean during 1992–2000 and a resultant slowdown of the shallow meridional overturning circulation in this Ocean. The estimated rate of the slowdown of this circulation, 6.8 ± 1.4 × 106 m3/s over the 9‐year period, is nearly 70% of the mean strength of this circulation. Such a change has important implications to upper‐ocean heat content and decadal climate variability of the Indian Ocean, as well as to the ecosystem and air‐sea exchange of CO2 in the region.
Response of the atmospheric boundary layer to a mesoscale oceanic eddy in the northeast Atlantic
Authors: Bourras Denis , Reverdin Gilles , Giordani Hervé , Caniaux Guy
Published: September 2004

Fields of air‐sea turbulent fluxes and bulk variables were derived from satellite sensor data from February to April 2001, over a region of the northeast Atlantic where a field experiment, Programme Océan Multidisciplinaire Meso Echelle (POMME), was conducted. The satellite products are in good agreement with in situ data in terms of heat fluxes, sea surface temperature, and wind speed. The central part of the experimental domain presented a cyclonic eddy in the ocean, which corresponded to a cold sea surface temperature (SST) anomaly. Winds were weaker within the eddy than outside of it, with lower latent and sensible heat loss. In order to analyze the relationship between the SST and wind anomalies, three numerical experiments were conducted with a regional atmospheric model. Three 3‐month runs of the model were performed, using a realistic SST field, a smoothed SST field in which the cold SST was not present (reference run), and an SST field where the cold anomaly was increased by two degrees, successively. The fields simulated with the realistic SST were consistent with satellite sensor derived observations. In particular, the weak wind area over the cold SST anomaly was successfully rendered, whereas it was not present in the forcing fields. Taken individually, the three runs did not reveal the presence of secondary circulations. However, anomalous secondary circulations were clearly identified with respect to the reference run. The origin of the latter circulations was investigated with the Giordani and Planton generalization of the Sawyer‐Eliassen equations. According to our results, differential heating induced by the cold SST anomaly mostly altered the vertical wind through the effect of friction and only marginally through pressure gradient forces. In the upper part of the boundary layer, the wind speed increased (decreased) over (downstream) the cold SST. We found that stability was the main factor that induced the simulated patterns of the friction term in the diagnostic equations. Therefore our results show that mesoscale wind patterns were significantly affected by SST gradients through the effect of stability, in a region of low oceanic eddy activity.
SNM‐dependent recombinational repair of oxidatively induced DNA damage in Arabidopsis thaliana
Authors: Molinier Jean , Stamm Marie‐Eve , Hohn Barbara
Published: September 2004

Two different roles for SNM (sensitive to nitrogen mustard) proteins have already been described: the SNM1/PSO2‐related proteins are involved in the repair of the interstrand crosslink (ICL) and the ARTEMIS proteins are involved in the V(D)J recombination process. Our study shows that an Arabidopsis SNM protein, although structurally closer to the SNM1/PSO2 members, shares some properties with ARTEMIS but also has novel characteristics. Arabidopsis plants defective for the expression of AtSNM1 did not show hypersensitivity to the ICL‐forming agents but to the chemotherapeutic agent bleomycin and to H2O2. AtSNM1 mutant plants are delayed in the repair of oxidative damage and did not show enhancement of the frequency of somatic homologous recombination on exposure to H2O2 and to the bacterial elicitor flagellin, both inducing oxidative stress, as observed in the control plants. Therefore, our results suggest the existence, in plants, of a novel SNM‐dependent recombinational repair process of oxidatively induced DNA damage.
Bubble‐induced detachment of affinity‐adsorbed erythrocytes
Authors: Barkley Suzanne , Johnson Harvey , Eisenthal Robert , Hubble John
Published: September 2004

It is desirable that cells adsorbed in affinity‐separation processes be easily recovered from the adsorption surface, without excessive dilution, once contaminants have been removed. The present study investigates the use of gas‐bubble‐induced shear stress for the recovery of affinity‐adsorbed human erythrocytes. This method has previously been demonstrated to be effective with yeast cells, where it allows cells to be attached, washed and detached under isocratic conditions. Concanavalin A (Con A), used as the binding agent, was attached to the inside of nylon tubes. Whole blood solution, diluted to an erythrocyte concentration of 1×108·ml−1 with PBS, was incubated with the Con A–nylon surface and then washed with PBS prior to elution. To effect elution, air bubbles of known volume were introduced to the buffer feed to the tubes and the effects of bubble size, bubble volume and bubble velocity on detachment being determined. The results obtained showed that the most significant parameter was bubble number, with up to 90% of attached cells being recovered using a five‐bubble sequence. Microscopic examination showed no evidence of mechanical damage to the detached cells.
Novozym 435‐catalysed transesterification of crude soya bean oils for biodiesel production in a solvent‐free medium
Authors: Du Wei , Xu Yuan‐yuan , Zeng Jing , Liu De‐hua
Published: September 2004

When crude soya‐bean oil was used as the source for biodiesel production, the yield of methyl ester was significantly lower than that from refined soya‐bean oil. The major difference between refined and crude soya‐bean oils was found to be due to the contents of phospholipids, free acid and water, which have varied influences on biodiesel production. Phospholipid content was the most influential factor; the higher the phospholipid content in the oil, the lower is the methyl ester yield. The optimal water activity was found to be in the range 0.12–0.44, and free acid included in crude soya‐bean oils showed no negative effect on enzymic transesterification. During our study of three‐step methanolysis of crude soya‐bean oils for biodiesel production, we noted that the second‐step methanolysis was much faster than the first‐step reaction. On the basis of this finding, the proposal that immersing pretreatment of lipase in oils contributes to improvement in enzyme activity has been put forward. In addition, it has been demonstrated that immersing pretreatment of lipase in oils could improve both the reaction rate and methyl ester yield significantly. A methyl ester yield of 94% could be achieved by immersing lipase in crude oils for 120 h and this was just as high as the yield for refined oils.
Herpes simplex virus VP22–human papillomavirus E2 fusion proteins produced in mammalian or bacterial cells enter mammalian cells and induce apoptotic cell death
Authors: Roeder Geraldine E. , Parish Joanna L. , Stern Peter L. , Gaston Kevin
Published: September 2004

Infection by high‐risk HPV (human papillomavirus) is supposed to be the primary cause of cervical cancer. The HPV E2 protein (E2) is a DNA‐binding protein that regulates viral gene expression and is required for efficient viral replication. Overexpression of the E2 protein in cervical cancer cells can induce growth arrest and/or apoptotic cell death, suggesting that E2 might be useful in the treatment of this disease. In the present study, we show that VP22 (herpes simplex virus VP22 protein) can be used to deliver E2 to target cells. VP22–E2 fusion proteins induce apoptosis in transiently transfected HPV‐transformed cervical carcinoma cell lines. However, VP22–E2 fusion proteins do not kill COS‐7 cells, probably because these cells constitutively express the simian‐virus‐40 T antigen and this protein sequesters the tumour suppressor protein p53. When COS‐7 cells producing VP22–E2 are seeded into cultures of HPV‐transformed cells, VP22–E2 enters the non‐producing cells and induces apoptosis. VP22–E2 proteins produced in bacterial cells can also enter cervical cancer cells and induce apoptosis in a dose‐dependent manner. Our results suggest that local delivery of VP22–E2 fusion proteins could be used to treat cervical cancer and other HPV‐associated diseases.
Generation of polyclonal antibodies against multiple proteins in a single rabbit and subsequent isolation of the specific immunoglobulins as a tool for proteomics research
Authors: Jacquot Jean‐Pierre , Rouhier Nicolas , Navrot Nicolas , Decottignies Jean‐Pierre
Published: September 2004

A single rabbit has been immunized with a mixture of six different proteins: green fluorescent protein, ferredoxin:NADP oxidoreductase, NADPH:thioredoxin reductase, thioredoxin f, fructose bisphosphatase and cyclophilin. Individual IgGs were re‐purified from the serum through specific interaction on small columns generated by grafting the antigen on a CNBr‐Sepharose matrix. The specificity of the isolated IgG was checked by Western‐blot analysis. Purified and specific IgG fractions were obtained with all antigens except thioredoxin f.
Overall volumetric oxygen transfer coefficient in an aerated bench‐top stirred fermenter in aqueous dispersions of sodium alginate
Authors: Parente Eugenio , Piraino Paolo , Fidaleo Marcello , Moresi Mauro
Published: September 2004

The dynamic gassing‐out method was used to assess the effect of stirrer speed, air flow rate and alginate concentration on the overall volumetric oxygen mass transfer coefficient (KLa) in a bench‐top stirred fermenter equipped with two Rushton‐type turbines. As the alginate concentration in the medium was increased from 0 to 2% (w/v), the liquid film time constant (τF) increased from approx. 30 to 300% of the electrode time constant (τE), clearly showing that the liquid‐probe diffusion film affected the oxygen‐electrode response. By accounting for delayed probe response, liquid film time constant and gas residence time, the corrected values of KLa were empirically correlated with the above independent variables as converted into gassed power input and aeration number per unit liquid volume and the effective liquid/water shear viscosity ratio. Despite an average error of 19%, such a correlation might be useful to adjust the oxygen transfer in bench‐top stirred reactors during viscous aerobic fermentations.
An efficient isocratic separation of hydroxycinnamates and their corresponding benzoates from microbial and plant sources by HPLC
Authors: Sachan Ashish , Ghosh Shashwati , Mitra Adinpunya
Published: September 2004

A rapid HPLC‐based separation method was developed to analyse phenolic flavour components. In the present study, ferulic acid, 4‐coumaric acid, 4‐hydroxybenzoic acid, 4‐hydroxybenzaldehyde, vanillic acid and vanillin were chromatographed on various C18 columns (Prodigy™ ODS2, Synergi™ Hydro‐RP, Lichrosorb® and Columbus™). A dual‐wavelength UV detector was used for the precise identification of the eluted components. An isocratic elution with aqueous trifluoroacetic acid (1 mM)/methanol (17:8) at a flow rate of 1.0 ml/min separated all the above six phenolic compounds within 21 min on a C18 reverse‐phase column (Synergi™ Hydro‐RP) with stable baseline resolution.
Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology
Authors: Ramnani Priya , Gupta Rani
Published: September 2004

A 3.5‐fold increase in keratinase production by Bacillus licheniformis RG1 was achieved by using statistical methods involving Plackett–Burman design and response surface methodology. Eight variables were screened using Plackett–Burman design. Of these, glucose, peptone and glutathione were found to affect the response signal positively, whereas CaCl2 had a negative effect. Further interaction of these factors, along with phosphate and incubation time, was studied using response surface methodology. An optimum keratinase production of 1295 units/mg dry weight was obtained with the following medium composition: 1% glucose, 1% peptone, 1% phosphate, 0.05% glutathione, 0.5% feather and 2% inoculum under shaking at 250 rev./min with an incubation period of 72 h at 37 °C. Keratinase production was found to be a function of biomass and maximum production occurred during the stationary phase.
Characterization of a calmodulin-regulated Ca2+-dependent-protein-kinase-related protein kinase, AtCRK1, from Arabidopsis
Authors: WANG Ying , LIANG Shuping , XIE Qi-Guang , LU Ying-Tang
Published: September 2004

An AtCRK1 [Arabidopsis thaliana CDPK (Ca2+-dependent protein kinase)-related protein kinase 1] has been characterized molecularly and biochemically. AtCRK1 contains the kinase catalytic domain and a CaM (calmodulin)-binding site. Our results demonstrated that AtCRK1 could bind CaM in a Ca2+-dependent manner. This kinase phosphorylated itself and substrates such as histone IIIS and syntide-2 in a Ca2+-independent manner and the activity was stimulated by several CaM isoforms through its CaM-binding domain. This domain was localized within a stretch of 39 amino acid residues at positions from 403 to 441 with Kd=67 nM for CaM binding. However, the stimulation amplification of the kinase activity of AtCRK1 by different CaM isoforms was similar.
Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family
Authors: WORMIT Alexandra , TRAUB Michaela , FLÖRCHINGER Martin , NEUHAUS H. Ekkehard , MÖHLMANN Torsten
Published: September 2004

Research on metabolism of nucleotides and their derivatives has gained increasing interest in the recent past. This includes de novo synthesis, analysis of salvage pathways, breakdown and transport of nucleotides, nucleosides and nucleobases. To perform a further step towards the analysis of nucleoside transport in Arabidopsis, we incubated leaf discs with various radioactively labelled nucleosides. Leaf cells imported labelled nucleosides and incorporated these compounds into RNA, but not into DNA. Furthermore, we report on the biochemical properties of three so far uncharacterized members of the Arabidopsis ENT (equilibrative nucleoside transporter) family (AtENT4, AtENT6 and AtENT7). After heterologous expression in yeast, all three proteins exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent Km values were in the range 3–94 μM, and transport was inhibited most strongly by deoxynucleosides, and to a smaller extent by nucleobases. Typical inhibitors of mammalian ENT proteins, such as dilazep and NBMPR (nitrobenzylmercaptopurine ribonucleoside, also known as nitrobenzylthioinosine) surprisingly exerted almost no effect on Arabidopsis ENT proteins. Transport mediated by the AtENT isoforms differed in pH-dependency, e.g. AtENT7 was not affected by changes in pH, AtENT3, 4 and 6 exhibited a less pronounced pH-dependency, and AtENT1 activity was clearly pH-dependent. Using a GFP (green fluorescent protein)-fusion protein transiently expressed in tobacco leaf protoplasts, a localization of AtENT6 in the plant plasma membrane has been revealed.
In brain mitochondria the branched-chain fatty acid phytanic acid impairs energy transduction and sensitizes for permeability transition
Authors: SCHÖNFELD Peter , KAHLERT Stefan , REISER Georg
Published: September 2004

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) accumulates at high levels throughout the body in the adult form of Refsum disease, a peroxisomal genetic disorder. However, it is still unclear why increased levels of phytanic acid have cytotoxic effects. In the present study, we examined the influence of non-esterified phytanic acid on energy-related functions of mitochondria from adult rat brain. Phytanic acid at low concentrations (5–20 μM, i.e. 5–20 nmol/mg of mitochondrial protein) de-energized mitochondria, as indicated by depolarization, stimulation of non-phosphorylating oxygen uptake and inhibition of the reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. The unbranched homologue palmitic acid exerted much smaller effects. In addition, phytanic acid reduced state 3 respiration, which was partly due to inhibition of the ADP/ATP carrier. Phytanic acid decreased the rate of adenine nucleotide exchange and increased the degree of control, which the ADP/ATP carrier has on state 3 respiration. Important for functional consequences is the finding that mitochondria, which are preloaded with small amounts of Ca2+ (100 nmol/mg of protein), became highly sensitized to rapid permeability transition even when only low concentrations of phytanic acid (below 5 μM) were applied. In conclusion, the incorporation of phytanic acid into the inner mitochondrial membrane increases the membrane H+ conductance and disturbs the protein-linked functions in energy coupling. This is most probably essential for the short-term toxicity of phytanic acid. Thus in neural tissue, which becomes enriched with phytanic acid, the reduction in mitochondrial ATP supply and the facilitation of the opening of the permeability transition pore are two major mechanisms by which the branched-chain fatty acid phytanic acid induces the onset of degenerative processes.
A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2
Authors: MAMPEL Jörg , MAIER Elke , TRALAU Tewes , RUFF Jürgen , BENZ Roland , COOK Alasdair M.
Published: September 2004

Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the inner membrane. tsaT was identified as a 1017-bp ORF (open reading frame) on plasmid pTSA upstream of the TSA-catabolic genes in the tsa operon. Expression of tsaT was regulated by TsaR, the transcriptional activator of the tsa regulon. The presence of tsaT was concomitant with the presence of the tsa operon in different TSA-degrading isolates. tsaT was expressed in Escherichia coli and was detected in the outer membrane. A 22-amino-acid leader peptide was identified. Purified protein reconstituted in lipid bilayer membranes formed anion-selective channels with a single-channel conductance of 3.5 nS in 1 M KCl. Downstream of tsaT, a constitutively expressed 720-bp ORF (tsaS) was identified. tsaS coded for a hydrophobic protein predicted to have six transmembrane helices and which is most likely localized in the cytoplasmic membrane. tsaS is adjacent to tsaT, but showed a different transcriptional profile.
Amino acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase
Authors: JUNG Sun-Mi , LE Dung Tien , YOON Sung-Sook , YOON Moon-Young , KIM Young Tae , CHOI Jung-Do
Published: September 2004

The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered Vmax values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.
Cargo- and compartment-selective endocytic scaffold proteins
Authors: SZYMKIEWICZ Iwona , SHUPLIAKOV Oleg , DIKIC Ivan
Published: September 2004

The endocytosis of membrane receptors is a complex and tightly controlled process that is essential for maintaining cellular homoeostasis. The removal of receptors from the cell surface can be constitutive or ligand-induced, and occurs in a clathrin-dependent or -independent manner. The recruitment of receptors into specialized membrane domains, the formation of vesicles and the trafficking of receptors together with their ligands within endocytic compartments are regulated by reversible protein modifications, and multiple protein–protein and protein–lipid interactions. Recent reports describe a variety of multidomain molecules that facilitate receptor endocytosis and function as platforms for the assembly of protein complexes. These scaffold proteins typically act in a cargo-specific manner, recognizing one or more receptor types, or function at the level of endocytic cellular microcompartments by controlling the movement of cargo molecules and linking endocytic machineries to signalling pathways. In the present review we summarize present knowledge on endocytic scaffold molecules and discuss their functions.
cAMP-response element modulator-τ activates a distinct promoter element for the expression of the phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene
Authors: TRAMER Federica , VETERE Amedeo , MARTINELLI Monica , PARONI Federico , MARSICH Eleonora , BOITANI Carla , SANDRI Gabriella , PANFILI Enrico
Published: September 2004

PHGPx (phospholipid hydroperoxide glutathione peroxidase) is a selenoprotein present in at least three isoforms in testis: cytosolic, mitochondrial and nuclear. All of these derive from the same gene and are structurally related with the exception of the snPHGPx (sperm nucleus-specific form), which differs from the others due to the presence of an arginine-rich N-terminus. It has been demonstrated recently that this N-terminus is encoded by an alternative exon located in the first intron of the PHGPx gene. The expression of snPHGPx has been attributed either to an alternative pre-mRNA splicing or to the presence of a distinct promoter region. Nevertheless, the exact molecular mechanism by which the expression of snPHGPx occurs has not been demonstrated so far. Preliminary sequence analysis of the region located upstream of the alternative exon revealed some potential DNA-binding sites, one of which is specific to the binding of CREM (cAMP-response element modulator) transcription factors. By using electrophoretic mobility-shift assays, we demonstrated that both nuclear protein extract from highly purified rat spermatid cells and recombinant CREM-τ protein can specifically bind to this element. Furthermore, we cloned a 1059 bp comprising the intron and the alternative exon for snPHGPx in the pCAT®3 reporter vector. By transient transfection experiments, we demonstrated that the expression of the transcription factor CREM-τ can induce the activation of the reporter gene in NIH-3T3 cell line. These results were confirmed by chromatin immunoprecipitation experiments performed on highly purified rat spermatid cells. On the basis of these results, we demonstrate that snPHGPx expression is mediated by the transcription factor CREM-τ, which acts as a cis-acting element localized in the first intron of the PHGPx gene.
Enhancing enhancers: new complexities in the retinoid regulation of gene expression
Authors: REDFERN Chris P. F.
Published: September 2004

Retinoic acid is a signalling molecule central to morphogenesis and musculoskeletal development. It can exist in several isomeric forms, of which all-trans- and 9-cis-retinoic acid are thought to be the most relevant as signalling molecules. Retinoic acid regulates gene expression via RARs (retinoic acid receptors) working as heterodimers with RXRs (retinoid X receptors). RXRs also heterodimerize with other nuclear receptors. In this issue of the Biochemical Journal, Harris et al. have shown that an enhancer responsible for chondrocyte-specific expression of the col11a2 gene is itself regulated by a retinoic-acid-dependent interaction with RXRβ bound to a downstream response element. Thus, RXRs bound to hormone-response elements can regulate gene expression indirectly via interactions with tissue-specific enhancers. This study raises interesting questions about the nature of the response element, the RXRβ partner and the ligands able to influence col11a2 expression, and will provide a model system with which to understand tissue and ligand specificity of retinoid responses.
Diminished L-arginine bioavailability in hypertension
Authors: MOSS Monique B. , BRUNINI Tatiana M. C. , SOARES de MOURA Roberto , NOVAES MALAGRIS Lúcia E. , ROBERTS Norman B. , ELLORY J. Clive , MANN Giovanni E. , MENDES RIBEIRO Antônio C.
Published: September 2004

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175±19 μmol/l) compared with control subjects (137±8 μmol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60±7 μmol·l−1·cells−1·h−1; n=16) compared with controls (90±17 μmol·l−1·cells−1·h−1; n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86±17 pmol·109 cells−1·min−1 in controls compared with 36±9 pmol·109 cells−1·min−1 in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12±0.02 pmol/108 cells; n=8) compared with controls (0.22±0.01 pmol/108 cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.