bluuebunny/crossref_metadata_2025 huggingface dataset:
VirE2, a Type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens
Authors: Atmakuri Krishnamohan , Ding Zhiyong , Christie Peter J.
Published: September 2003

Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens – a two‐hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) – and by immunoprecipitation of chemically cross‐linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2‐terminal membrane‐spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB‐encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T‐DNA transfer intermediate. Finally, VirE2 is recruited to polar‐localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector–coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.
Genome‐based analysis of virulence genes in a non‐biofilm‐forming Staphylococcus epidermidis strain (ATCC 12228)
Authors: Zhang Yue‐Qing , Ren Shuang‐Xi , Li Hua‐Lin , Wang Yong‐Xiang , Fu Gang , Yang Jian , Qin Zhi‐Qiang , Miao You‐Gang , Wang Wen‐Yi , Chen Run‐Sheng , Shen Yan , Chen Zhu , Yuan Zheng‐Hong , Zhao Guo‐Ping , Qu Di , Danchin Antoine , Wen Yu‐Mei
Published: September 2003

Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non‐pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non‐biofilm forming, non‐infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from δ‐haemolysin and β‐haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.
Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgareL.)
Authors: KOLB K. J. , EVANS R. D.
Published: September 2003

The occurrence of nitrogen isotope discrimination with absorption and assimilation of nitrate (NO3–) and ammonium (NH4+) was investigated using two genotypes of barley,Hordeum vulgareL. cv. Steptoe and Az12 : Az70, the latter of which lacks the characterized nitrate reductase isozymes. Plants were grown under two situations: a closed system with limited nitrogen or an open system with unlimited nitrogen, to elucidate the conditions and processes that influence discrimination. There was no discrimination observed for Az12 : Az70 when supplied with limited nitrogen. Discrimination was observed for Steptoe seedlings at high external NO3–concentrations, but not with low NO3–when assimilation is probably rapid and complete. The same pattern was observed for Steptoe when NH4+was supplied; indicating that for both nitrogen forms discrimination is dependent upon the presence of the assimilatory enzyme and the external concentration. The implications of this study are that both internal (assimilatory enzyme distribution) and external (source concentration) factors may have a larger impact on tissueδ 15N than the form of nitrogen utilized. This suggests that tissueδ 15N may not always be a reliable indicator of a plant's integrated nitrogen nutrition.
Evaluation of models of leaf water 18O enrichment using measurements of spatial patterns of vein xylem water, leaf water and dry matter in maize leaves
Authors: GAN K. S. , WONG S. C. , YONG J. W. H. , FARQUHAR G. D.
Published: September 2003

The effectiveness of several leaf water models (‘string‐of‐lakes’, ‘desert river’ and the Farquhar–Gan model) are evaluated in predicting the enrichment of leaf water along a maize leaf at different humidities. Progressive enrichment of both vein xylem water and leaf water was observed along the blade. At the tip, the maximum observed enrichment for the vein water was 17.6‰ at 50% relative humidity (RH) whereas that for the leaf water was 50‰ at 34% RH and 19‰ at 75% RH. The observed leaf water maximum was a fraction (0.5–0.6) of the theoretically possible maximum. The ‘string‐of‐lakes’ and ‘desert river’ models predict well the variation of leaf water enrichment pattern with humidity but overestimate the average enrichment of bulk leaf water. However, the Farquhar–Gan model gives good prediction for these two aspects of leaf water enrichment. Using the anatomical dimensions of vein xylem overestimates the effective longitudinal Péclet number (Pl). Possible explanations for this discrepancy between the effective and the xylem‐based estimate of Pl are discussed. The need to characterize the heterogeneity of transpiration rate over the leaf surface in studies of leaf water enrichment is emphasized. The possibility that past atmospheric humidity can be predicted from the slope of the Δ18O spatial variation of leaf macrofossils found in middens is proposed.
Molecular identification of proline‐rich protein genes induced during root formation in grape (Vitis vinifera L.) stem cuttings
Authors: THOMAS P. , LEE M. M. , SCHIEFELBEIN J.
Published: September 2003

Vegetative reproduction relies on the initiation of new plant organs in response to environmental changes. The rapid formation of roots, and ultimately whole plants, from stem cuttings of grape (Vitis vinifera L.) provides a useful system to investigate the physiological and molecular basis of organ initiation during vegetative reproduction. In the present study the differential RNA display technique was employed to identify two genes, VvPRP1 and VvPRP2, that are induced in stem cuttings of grape during rooting. Each of these genes encodes a distinct type of proline‐rich protein that is related to different groups of putative cell wall proteins, and their expression is rapidly induced in stem segments within 6 h after severing. Further, each gene's transcript becomes most concentrated in the basal portion of the stem segment in the region of new root formation. Induction of these genes is not significantly enhanced by indole‐3‐acetic acid (IAA) treatment, and the expression of the VvPRP1 gene, but not the VvPRP2 gene, is wound‐inducible. These results suggest that these VvPRP genes play an important role in the initiation of new roots on grape stem cuttings, perhaps by altering the cell wall mechanical properties to enable root emergence.
Electrophysiological localization of blue light sensory sites in etiolated dicotyledon seedlings
Authors: BABOURINA O. K. , NEWMAN I. A. , SHABALA S. N.
Published: September 2003

Changes induced by blue light (BL) in ion fluxes and pH in etiolated seedlings of dicotyledons (Cucumis sativus L., Phaseolus lunatus L., Pisum sativum L.) were studied using a non‐invasive ion‐selective microelectrode technique (MIFE). BL induced significant Ca2+ uptake within the first 5 min after onset in upper parts of the whole cucumber seedling. To locate the sites of the BL perception, studies on excised segments were performed. The data obtained demonstrated that the observed Ca2+ uptake is associated with the very top of the hypocotyl hook of cucumber and butter bean, and with the whole epicotyl of pea, although the response was stronger for the upper parts of the epicotyl. Addition of Gd3+ and La3+ into the bathing solution completely prevented Ca2+ influx, implicating the involvement of Ca2+ channels in the regulation of Ca2+ transport during BL exposure. BL‐induced changes in H+ and Ca2+ fluxes were not likely to be linked to the one mechanism, as they were separated spatially and pharmacologically.
Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots
Authors: WASAKI J. , YONETANI R. , KURODA S. , SHINANO T. , YAZAKI J. , FUJII F. , SHIMBO K. , YAMAMOTO K. , SAKATA K. , SASAKI T. , KISHIMOTO N. , KIKUCHI S. , YAMAGISHI M. , OSAKI M.
Published: September 2003

As most soil phosphates exist as insoluble inorganic phosphate and organic phosphates, higher plants have developed several strategies for adaptation to low phosphorus (P). These include the secretion of acid phosphatase and organic acids, induction of the inorganic phosphate (Pi) transporter and the substitution of some enzyme activities as alternative pathways to increase P utilization efficiency. It has been proposed that plants also have a ‘pho regulon’ system, as observed in yeast and Escherichia coli; however, the detail of the regulation system for gene expression on P status is still unclear in plants. To investigate the alteration of gene expression of rice roots grown under P‐deficient conditions, a transcriptomic analysis was conducted using a cDNA microarray on rice. Based on the changes of gene expression under a –P treatment, the up‐regulation of some genes due to P deficiency was confirmed . Some new important metabolic changes are suggested, namely: (1) acceleration of carbon supply for organic acid synthesis through glycolysis; (2) alteration of lipid metabolism; (3) rearrangement of compounds for cell wall; and (4) changes of gene expression related to the response for metallic elements such as Al, Fe and Zn.
Influence of elevated CO2 and nitrogen nutrition on photosynthesis and nitrate photo‐assimilation in maize (Zea mays L.)
Authors: COUSINS A. B. , BLOOM A. J.
Published: September 2003

Measurements of CO2 and O2 gas exchange and chlorophyll a fluorescence were used to test the hypothesis that elevated atmospheric CO2 inhibits nitrate (NO3–) photo‐assimilation in the C4 plant, maize (Zea mays L.). The assimilatory quotient (AQ), the ratio of net CO2 assimilation to net O2 evolution, decreases as NO3– photo‐assimilation increases so that the difference in AQ between the ammonium‐ and nitrate‐fed plants (ΔAQ) provided an in planta estimate of NO3– photo‐assimilation. In fully expanded maize leaves, NO3– photo‐assimilation was detectable only under high light and was not affected by CO2 treatments. Furthermore, CO2 assimilation and O2 evolution were higher under NO3– than ammonia (NH4+) regardless of CO2 levels. In conclusion, NO3– photo‐assimilation in maize primarily occurred at high light when reducing equivalents were presumably not limiting. Nitrate photo‐assimilation enhanced C4 photosynthesis, and in contrast to C3 plants, elevated CO2 did not inhibit foliar NO3– photo‐assimilation.
Reactive oxygen species stimulate homologous recombination in plants
Authors: KOVALCHUK I. , FILKOWSKI J. , SMITH K. , KOVALCHUK O.
Published: September 2003

Coping with the continuous production of free radicals is a daily routine of the cell. Despite their toxicity, the reactive oxygen species (ROS) are involved in dual physiological action – signal transduction and immune response. We analysed the influence of oxidative stress‐generating compounds, rose Bengal (RB), paraquat (PQ) and amino‐triazole (ATZ) on the genome stability of Arabidopsis using transgenic recombination‐monitoring plants. Homologous recombination frequencies in plants were increased upon the treatment with RB and PQ but not ATZ. Application of the N‐acetyl‐L‐cysteine (NAC), radicals scavenging compound, decreased the DNA damage caused by RB. Interestingly, the incubation of plants with very low concentration of RB (less than 0.2 µM) led to the subsequent increase in plant tolerance to methyl methane sulfonate (MMS): stronger plants with a lower increase of homologous recombination frequency. In contrast, the incubation of plants with 0.5 µM of RB resulted in the potentiation of the MMS effect: the weaker plants with higher frequency of recombination. The data of the present study suggest the existence of a dual concentration‐dependent role of ROS in plants.
Signal transduction downstream of salicylic and jasmonic acid in herbivory‐induced parasitoid attraction by Arabidopsis is independent of JAR1 and NPR1
Authors: VAN POECKE R. M. P. , DICKE M.
Published: September 2003

Plants can defend themselves indirectly against herbivores by emitting a volatile blend upon herbivory that attracts the natural enemies of these herbivores, either predators or parasitoids. Although signal transduction in plants from herbivory to induced volatile production depends on jasmonic acid (JA) and salicylic acid (SA), the pathways downstream of JA and SA are unknown. Use of Arabidopsis provides a unique possibility to study signal transduction by use of signalling mutants, which so far has not been exploited in studies on indirect plant defence. In the present study it was demonstrated that jar1‐1 and npr1‐1 mutants are not affected in caterpillar (Pieris rapae)‐induced attraction of the parasitoid Cotesia rubecula. Both JAR1 and NPR1 (also known as NIM1) are involved in signalling downstream of JA in induced defence against pathogens such as induced systemic resistance (ISR). NPR1 is also involved in signalling downstream of SA in defence against pathogens such as systemic acquired resistance (SAR). These results demonstrate that signalling downstream of JA and SA differs between induced indirect defence against herbivores and defence against pathogens such as SAR and ISR. Furthermore, it was demonstrated that herbivore‐derived elicitors are involved in induced attraction of the parasitoid Cotesia rubecula
Regulation of nitrogen uptake by Fagus sylvatica on a whole plant level – interactions between cytokinins and soluble N compounds
Authors: COLLIER M. D. , FOTELLI M. N. , NAHM M. , KOPRIVA S. , RENNENBERG H. , HANKE D. E. , GEßLER A.
Published: September 2003

Pedospheric nitrate uptake is a strongly regulated process adapted to the N demand of the whole plant. Pre‐requisites for an integrating regulatory system are signal substances communicating the N demand of the shoot to the roots. In the current study it was shown that an additional atmospheric N source results in activation of cytokinins in the shoot as indicated by an increase in the trans‐hydroxylation ratio, and in increased shoot‐to‐root transport of cytokinins in the phloem. Phloem mobility and basipetal transport of cytokinins were also demonstrated by feeding zeatin riboside into the phloem. The resulting enrichment of cytokinins in the roots caused an increased expression of a high‐affinity nitrate transporter, the enrichment of amino compounds (Glu, Val, Phe, Lys) in the fine roots and a significant decrease in nitrate net uptake. Significant enrichment in amino acids – accompanied by decreased nitrate net uptake but not by increased expression of the high‐affinity nitrate transporter – was also observed when Gln the major long‐distance transport form of nitrogen in beech was fed into the phloem. These results provide experimental evidence that, in addition to amino compounds, cytokinins that are known to cycle within the plant are also communicating changes in N metabolism from the shoot to the roots. Apparently increased cytokinin contents in the root can transiently increase nitrate uptake, but this up‐regulation may be overridden by transcriptional and post‐transcriptional down‐regulation mediated by amino compounds.
Scaling phloem transport: water potential equilibrium and osmoregulatory flow
Authors: THOMPSON M. V. , HOLBROOK N. M.
Published: September 2003

In this work, the common assumption that phloem sap is in water potential equilibrium with the surrounding apoplast was examined. With a dimensionless model of phloem translocation that scales with just two dimensionless parameters (R̂and F̂), a ‘map’ of phloem behaviour as a function of these parameters was produced, which shows that the water potential equilibrium assumption (R̂F̂ >> 1) is valid for essentially all realistic values of the relevant scales. When in water potential equilibrium, a further parameter reduction is possible that limits model dependence to a single parameter (F̂), which describes the ratio of the solution's osmotic strength to its axial pressure drop. Due to the locally autonomous nature of individual sieve element/companion cell complexes, it is argued that long‐distance integrative control is most efficient when F̂ is large (that is, when the pressure drop is relatively small), permitting the sieve tube to regulate solute loading in response to global changes in turgor. This mode of transport has been called ‘osmoregulatory flow.’ Limitations on the pressure drop within the transport phloem could require that sieve tubes be shorter than the long axis of the plant, and thus arranged in series and hydraulically isolated from one another.
Regulation of T Helper Cell Differentiation In Vivo by GP43 from Paracoccidioides brasiliensis Provided by Different Antigen‐Presenting Cells
Authors: Ferreira K. S. , Lopes J. D. , Almeida S. R.
Published: September 2003

Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve into different clinical forms that are associated with various degrees of suppressed cell‐mediated immunity. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated whether the nature of antigen‐presenting cells (APCs) can influence the Th1/Th2 balance in vivo. It was observed that the injection of mature dendritic cells (DCs), macrophages and B cells primed the mice and induced a proliferation of T cells in vitro. It was seen that DCs from resistant mice stimulated predominantly interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ), whereas macrophages activated IL‐10, IL‐4 and IFN‐γ‐secreting T cells and B cells IL‐4 and IL‐10 only. Results presented here clearly demonstrate that DC drives the development of cells secreting Th1‐derived cytokines, whereas B cells induce the differentiation of a Th2 phenotype in vivo.
Generation of Antibodies to the Urokinase Receptor (uPAR) by DNA Immunization of uPAR Knockout Mice: Membrane‐Bound uPAR is Not Required for an Antibody Response
Authors: Pass J. , Gårdsvoll H. , Lund L. R. , Danø K. , Høyer‐Hansen G.
Published: September 2003

The urokinase receptor (uPAR) is a glycolipid‐anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes including cancer invasion. Furthermore, uPAR is found on the surface of both dendritic cells (DCs) and T cells, and has been proposed to play a role in DC‐induced T‐cell activation and, therefore, in the induction of an immune response. In order to investigate the possibility of using DNA immunization for the generation of poly‐ and monoclonal antibodies to uPAR, we injected wild‐type mice and mice deficient in uPAR (uPAR knockouts) intramuscularly with plasmid DNA encoding a carboxy‐terminal truncated soluble form of the human uPAR. Multiple injections of 100 µg of DNA resulted in a strong and specific antibody response in all mice irrespective of genotype. Antisera with a maximum titre of 32,000 were obtained, comparable with that obtained after immunization with recombinant uPAR. The subclass distribution of uPAR‐specific antibodies in the sera demonstrated the induction of a mixed TH1/TH2 response, irrespective of the genotype of the mice. Our results demonstrate the possibility of generating high titre antibodies to uPAR by DNA immunization of wild‐type as well as uPAR knockout mice, and that cell surface uPAR is not indispensable for the generation of a humoral immune response.
The Stimulus‐Dependent Release of Eosinophil Cationic Protein and Eosinophil Protein X Increases in Apoptotic Eosinophils
Authors: Seton K. , Håkansson L. , Karawajczyk M. , Venge P.
Published: September 2003

Apoptotic cells are regarded as inert bodies that turn off intracellular processes and functional abilities. To study the changes in the ability of eosinophils to release their granule proteins while undergoing apoptosis. Eosinophils were cultured for up to 72 h. Living cells were separated from the apoptotic cells and their release of eosinophil cationic protein (ECP) and eosinophil protein X (EPX) was measured in response to serum‐opsonized sephadex particles and phorbol 12‐myristate 12‐acetate (PMA). Changes in cell structure were examined by electron microscopy, and surface receptor expression of β1‐ and β2‐integrins was investigated by flow cytometry. Stimulus‐dependent release of the granule proteins ECP and EPX was found to increase in apoptotic eosinophils, whereas surface expression of β1‐ and β2‐integrins was downregulated. Ultrastructural examination revealed that the granules of apoptotic eosinophils were translocated to the periphery of the cell, just beneath the plasma membrane. Apoptotic eosinophils are able to release their toxic granule proteins, which is probably because of the rearrangement of the cytoskeleton and spontaneous translocation of granules to the membrane. Our results suggest that apoptotic eosinophils are potentially harmful cells that have retained their ability to react to certain extracellular stimuli. The findings point to unexpected consequences of eosinophil apoptosis.
Histamine Inhibits Neutrophil NADPH Oxidase Activity Triggered by the Lipoxin A4 Receptor‐Specific Peptide Agonist Trp‐Lys‐Tyr‐Met‐Val‐Met
Authors: Betten Å. , Dahlgren C. , Hermodsson S. , Hellstrand K.
Published: September 2003

The vasoactive amine histamine is found at high concentrations in the immune and inflammatory tissues. Earlier studies have revealed that histamine regulates the nicotinamide‐adenine dinucleotide phosphate (NADPH) oxidase‐dependent formation of oxygen radicals by phagocytic cells. However, the effects of histamine on intracellular signal transduction mechanisms of relevance to oxidase regulation remain controversial. For this study, we investigated the effects of histamine on NADPH oxidase activity in human neutrophil granulocytes triggered by a lipoxin A4 receptor agonist [the hexapeptide Trp‐Lys‐Tyr‐Met‐Val‐Met (WKYMVM), a formyl peptide receptor (FPR) agonist (the chemotactic tripeptide formylmethionyl‐leucyl‐phenylalanine (fMLF)) and an activator of protein kinase C (phorbol myristate acetate (PMA)]. We report that histamine, acting via H2‐type histamine receptors (H2R), suppresses NADPH oxidase‐dependent formation of oxygen radicals induced by WKYMVM and fMLF but not that induced by PMA. Peptide‐induced mobilization of granule‐localized complement receptor 3 (CR3) was unaffected by histamine suggesting that the inhibition specifically affected NADPH oxidase activation. Our data suggest that histamine downregulates FPRL1‐ and FPR‐induced NADPH oxidase activity upstream of protein kinase C (PKC) and downstream of the separation of the peptide‐induced signal into granule secretion and oxidase activation.
Severe Acute Respiratory Syndrome – a New Coronavirus from the Chinese Dragon's Lair*
Authors: Knudsen T. B. , Kledal T. N. , Andersen O. , Eugen‐Olsen J. , Kristiansen T. B.
Published: September 2003

The recent identification of a novel clinical entity, the severe acute respiratory syndrome (SARS), the rapid subsequent spread and case fatality rates of 14–15% have prompted a massive international collaborative investigation facilitated by a network of laboratories established by the World Health Organization (WHO). As SARS has the potential of becoming the first pandemic of the new millennium, a global warning by the WHO was issued on 12 March 2003. The disease, which is believed to have its origin in the Chinese Guangdong province, spread from Hong Kong via international airports to its current worldwide distribution. The concerted efforts of a globally united scientific community have led to the independent isolation and identification of a novel coronavirus from SARS patients by several groups. The extraordinarily rapid isolation of a causative agent of this newly emerged infectious disease constitutes an unprecedented scientific achievement. The main scope of the article is to provide the clinician with an overview of the natural history, epidemiology and clinical characteristics of SARS. On the basis of the recently published viral genome and structural features common to the members of the coronavirus family, a model for host cell–virus interaction and possible targets for antiviral drugs are presented. The epidemiological consequences of introducing a novel pathogen in a previously unexposed population and the origin and evolution of a new and more pathogenic strain of coronavirus are discussed.
Enhanced Activation of CD83‐Positive T Cells*
Authors: Wolenski M. , Cramer S. O. , Ehrlich S. , Steeg C. , Fleischer B. , Von Bonin A.
Published: September 2003

CD83 is a marker molecule for mature dendritic cells (DCs) but is also substantially expressed on activated T cells in humans and mice. Its function is unknown, but CD83 knockout mice show an impaired thymic maturation of CD4‐positive cells and soluble CD83 inhibits partially antigen‐specific responses in vitro pointing to a role of CD83 in the immune system. Here we show that CD83‐positive T cells produce strongly increased amounts of interferon‐γ and interleukin‐2. In contrast, constitutive expression of CD83 on DCs alters neither the activation of DCs following addition of lipopolysaccharide nor the ability to present antigenic peptides. Thus, the expression of CD83 on T cells has direct functional consequences for tuning the activation threshold.
In Situ Expression of CD40, CD40L (CD154), IL‐12, TNF‐α, IFN‐γ and TGF‐β1 in Murine Lungs during Slowly Progressive Primary Tuberculosis
Authors: Mogga S. J. , Mustafa T. , Sviland L. , Nilsen R.
Published: September 2003

The distribution and expression of CD40, its ligand CD40L (154) and related cytokines interleukin‐12 (IL‐12), tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) and transforming growth factor‐β1 (TGF‐β1) were studied in the lungs of B6D2F1 hybrid mice during slowly progressive primary tuberculosis (TB) by immunohistochemistry. CD40 and CD40L are implicated in cell‐mediated immunity (CMI) causing activation or apoptosis of infected cells. The phenomenon of apoptosis is associated with Mycobacterium tuberculosis survival. In this study, using frozen lung sections (n = 33), our results showed increased CD40, IL‐12 and TGF‐β1 expression in macrophages with progression of disease. High percentages of mycobacterial antigens (M.Ags), CD40L and IFN‐γ expression were maintained throughout infection, and TNF‐α‐expressing cells were decreased. In lymphocytes, the percentage of IFN‐γ‐positive cells was increased, but CD40L and IL‐12 were maintained with the progression of disease. M.Ags, CD40 and CD40L were expressed in the same areas of the lesions. We conclude that changes in the expression of CD40–CD40L and cytokines associated with M. tuberculosis infection favour the hypothesis that M. tuberculosis causes resistance of host cells to apoptosis causing perpetuation of infection.
The Cholera Toxin B Subunit Directly Costimulates Antigen‐Primed CD4+ T Cells Ex Vivo
Authors: Wang M. , Bregenholt S. , Petersen J. S.
Published: September 2003

The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination. However, the mechanisms behind its adjuvant effects remain unclear. Here, we have used an ex vivo system to elucidate these mechanisms in antigen‐specific T cells. Using splenocytes from keyhole limpet haemocyanin (KLH)‐immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH‐specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier. However, rCTB strongly enhanced the KLH‐specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior. This adjuvant effect was dose‐dependent, with maximum at 30–300 ng/ml rCTB. At higher doses of CTB this effect declined because of the induction of apoptosis. Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH‐specific CD4+ and CD8+ T‐cell proliferation but not B cells. Enzyme‐linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH‐specific CD4+ T‐cell‐mediated production of interleukin‐2 (IL‐2), IL‐4 and interferon‐γ(IFN‐γ) by four to fivefold. Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e. rCTB mediated a twofold increase in the ex vivo T‐cell response when used as a classical adjuvant in a secondary, but not in a primary KLH‐immunization regimen. Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen‐primed CD4+ and CD8+ T cell in a dose‐dependent manner. This new insight might be valuable in the future rational design of bacterial toxin‐based vaccines.
Selective Response of Dermal Th‐1 Cells to 20–50 kDa Streptococcal Cell‐Wall Proteins in Chronic Plaque Psoriasis
Authors: Baker B. S. , Ovigne J.‐M. , Fischetti V. A. , Powles A. , Fry L.
Published: September 2003

We have recently described a dermal Th‐1 subset in skin lesions of psoriasis which recognizes cell‐wall extract isolated from group A streptococci (GAS). As a first step in the identification of the streptococcal proteins involved, dermal T‐cell lines (TCL) cultured from the lesional skin of 12 human leucocyte antigen (HLA)‐typed psoriasis patients were stimulated with GAS cell‐wall extract and 14 fractions (MWt approximately 20–100 kDa) separated from the cell‐wall extract by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electroelution, stained for intracellular interferon‐γ(IFN‐γ) expression and analysed by flow cytometry. All the TCL responded to GAS cell‐wall extract to varying extents (3.5–27.6% IFN‐γ+). This response was consistently directed against 20–50 kDa cell‐wall fractions and inhibited by anti‐HLA‐DR antibody. TCL with higher responses to GAS cell‐wall extract recognized a larger number of fractions within this range than the lower responder TCL. No difference between the level and pattern of response to the fractions was observed for TCL from HLA‐DR7+ (n = 6) and HLA‐DR7– (n = 6) individuals. This preliminary study has shown a selective response to lower MWt proteins expressed on GAS cell wall by skin Th‐1 cells in psoriasis. Further studies are required to identify the proteins involved.
T Helper 2 Immunity to Hepatitis B Surface Antigen Primed by Gene‐Gun‐Mediated DNA Vaccination can be Shifted Towards T helper 1 Immunity by Codelivery of CpG Motif‐Containing Oligodeoxynucleotides
Authors: Zhou X. , Zheng L. , Liu L. , Xiang L. , Yuan Z.
Published: September 2003

Gene‐gun‐mediated DNA immunization usually induces predominant T helper 2 (Th2) type immune response. As oligodeoxynucleotides (ODN)‐containing unmethylated CpG motifs can activate the innate immune system in a Th1‐biased way, the potential of codelivery of CpG motifs‐containing ODN (CpG‐ODN) with plasmid DNA to switch the gene‐gun‐mediated Th2 immune response was evaluated in this study. Here we show that codelivery of CpG‐ODN with plasmid DNA at certain ratio (10/1) can enhance the Th1 humoral and cell‐mediated immune responses in gene‐gun‐mediated DNA immunization in BALB/c mice, including increasing the hepatitis B surface antigen‐specific total immunoglobulin G (IgG), IgG2a subclass, cytotoxic T‐cell lymphocyte activity as well as interferon‐γ (IFN‐γ) secretion. Taken together, these results demonstrate that codelivery of CpG‐ODN with recombinant plasmid DNA by gene gun can shift the gene‐gun‐mediated DNA immune response from Th2 towards Th1.
Syngeneic Pregnancy Induces Overexpression of Natural Killer T Cells in Maternal Liver
Authors: Mrakovčić‐Šutić I. , Šimin M. , Radić D. , Rukavina D. , Radošević‐Stašić B.
Published: September 2003

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self‐reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines.

To elucidate whether some of these changes occur even during the syngeneic pregnancy, we made phenotypic and functional characterization of mononuclear lymphatic cells (MNLCs) isolated from the liver and spleen of pregnant C57BL/6 mice, testing their cytotoxicity against syngeneic thymocytes as well as against NK‐ and lymphokine‐activated killer (LAK)‐sensitive targets. The data have shown that on the sixteenth day of syngeneic pregnancy TCRint, NK1.1+ and IL‐2Rβ+ cells were accumulated in the liver, while the quantities of CD4+ and CD8+ T cells and total number classical NK (NK1.1+CD3– or IL‐2Rβ+CD3–) cells were increased in the spleen. Pregnancy‐activated hepatic and splenic MNLCs were more cytotoxic against syngeneic thymocytes, YAC‐1 and P815 targets, suggesting that the maternal liver is a main producer of autoreactive NKT clones, which subsequently augment NK‐ and LAK cell‐mediated cytotoxicity in the liver and spleen.
Fusion of Interleukin‐2 to Subunit Antigens Increase their Antigenicity In Vitro Due to an Interleukin‐2 Receptor β‐Mediated Antigen Uptake Mechanism
Authors: Wales J. , Baird M. , Davies N. , Buchan G.
Published: September 2003

Subunit vaccines, based on one or more epitopes, offer advantages over whole vaccines in terms of safety but are less antigenic. We investigated whether fusion of the cytokine interleukin‐2 (IL‐2) to influenza‐derived subunit antigens could increase their antigenicity. The fusion of IL‐2 to the subunit antigens increased their antigenicity in vitro. Encapsulation of the subunit antigen in liposomes also increased its antigenicity in vitro, yet encapsulation of the subunit IL‐2 fusion did not. The use of anti‐IL‐2 receptor β (IL‐2Rβ) antibody to block the receptor subunit on macrophages suggested that the adjuvancy exerted by IL‐2 in our in vitro system is due to, at least in part, a previously unreported IL‐2Rβ‐mediated antigen uptake mechanism.
Natural Autoantibodies as Tools to Predict the Outcome of Immune Response?
Authors: Kohler H. , Bayry J. , Nicoletti A. , Kaveri S. V.
Published: September 2003

Natural autoantibodies (NAbs), produced by B‐1 B‐cells, are directed against autoantigens and pathogens. NAbs can capture and present antigen to T helper cells thereby initiating adaptive immunities. It is proposed that screening for NAbs against pathogens will predict the strength of an antigen‐induced immune response and could be used as a tool for vaccine development.
Hybridomas Expressing γδ T‐Cell Receptors Respond to Cardiolipin and β2‐Glycoprotein 1 (Apolipoprotein H)
Authors: Born W. K. , Vollmer M. , Reardon C. , Matsuura E. , Voelker D. R. , Giclas P. C. , O'Brien R. L.
Published: September 2003

Hybridomas expressing murine γδ T‐cell receptors were found to produce cytokines in response to cardiolipin (CL) and structurally related anionic phospholipids. This response required serum at concentrations related to the amount of CL in cultures. The purified serum factor, β2‐glycoprotein 1 (β2‐GP1) (apolipoprotein H), supported the CL response alone, whereas several other serum proteins and ovalbumin did not. β2‐GP1 is known to form complexes with anionic phospholipids, particularly CL, which are often recognized by pathological autoantibodies. We speculate that γδ T cells also recognize such complexes and that the hybridoma response reported here reflects this specificity.
Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes
Authors: Thompson W.F. , Beven A.F. , Wells B. , Shaw P.J.
Published: August 2003

Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and thus must be catalysed by RNA polymerase I. Immunofluorescence and confocal microscopy shows that incorporation occurs in a pattern consisting of many small foci distributed widely through the dense fibrillar component of the nucleoli. Immunogold labelling using silver‐enhanced Nanogold probe at the electron microscopic level confirms the sites of transcription as small foci approximately 200nm in diameter. Simultaneous fluorescence in situ hybridization with a probe to the external transcribed spacer (ETS) region of the pre‐rRNA shows that the structures revealed by this probe and the BrUTP immunofluorescence labelling are very similar. A probe to the transcribed portion of the rDNA (18S) also shows a good correlation to the sites of BrUTP incorporation within the nucleolus. On the other hand a probe to the non‐transcribed intergenic spacer region (NTS) shows very little coincidence with the sites of BrUTP incorporation, and double fluorescence in situ labelling with both 18S and NTS probes confirms this difference in localization. These results suggest that most BrUTP foci correspond to single transcribed genes.
Requirement of sense transcription for homology‐dependent virus resistance and trans‐inactivation
Authors: English James J. , Davenport Guy F. , Elmayan Taline , Vaucheret Hervé , Baulcombe David C.
Published: August 2003

Transgenic tobacco plants carrying post‐transcriptionally, silenced (L) or conditionally high‐expressing (Hc) cauliflower mosaic virus 35S promoter:β‐glucuronidase (GUS) transgenes were tested for their ability to support replication of a potato virus X derivative with an inserted GUS coding sequence (PVX.GUS). PVX.GUS accumulated to high levels on non‐transformed tobacco. In contrast, both L and Hc tobacco were resistant to PVX.GUS. Another line, 271, carries a 35S‐driven transgene that can down‐regulate transcription from any other 35S promoter in trans. When the 271 locus was introduced into the L and Hc lines, virus resistance was suppressed. The L tobacco line also trans‐inactivated transient GUS expression from an extra‐chromosomal T‐DNA delivered by Agrobacterium. This trans‐inactivation was not observed when the 271 locus was present. In addition, it was found that the 271 locus suppressed the virus resistance normally conferred by a 35S‐driven transgene derived from the PVX replicase open reading frame. These results indicate that sense transcription is required for a transgene to condition homology‐dependent virus resistance and post‐transcriptional trans‐inactivation.
Microdissection and amplification of coding sequences from a chromosome fragment restoring male fertility in alloplasmic male‐sterile tobacco.
Authors: Hernould Michel , Glimelius Kristina , Veuskens Jacky , Bergman Per , Mouras Armand
Published: August 2003

An alloplasmic male‐sterile line of tobacco, containing the nucleus of Nicotiana tabacum and the cytoplasm of Nicotiana repanda, is restored to fertility by introgression of an alien chromosome fragment obtained from the cytoplasm donor. To isolate the restorer gene(s), the alien chromosome fragment was microdissected from metaphase plates of the restored line. The microdissected chromosomes represented only 0.1 pg of DNA, which was amplified using a degenerate oligonucleotide‐primed PCR method (DOP–PCR), from which a chromosome fragment specific library was created. Compared with previous strategies used for microcloning, a modified and improved method was developed by the subsequent isolation of expressed sequences. The library was screened with cDNA probes synthesized by reverse transcription and DOP–PCR amplification (RT/DOP–PCR), of total RNAs isolated from early developing restored and male‐sterile flower buds. By this strategy, transcribed DNA sequences specific for the restored line were cloned.
cGMP and glutathione‐conjugate transport in human erythrocytes
Authors: Klokouzas Antonios , Wu Chung‐Pu , van Veen Hendrik W. , Barrand Margery A. , Hladky Stephen B.
Published: August 2003

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance‐associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR‐L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR‐L23/P cells. MRP4 is detected in MOR/R but not COR‐L23/R cells. Uptake of cGMP into inside‐out membrane vesicles prepared by a spontaneous, one‐step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 µm. This transport is reduced by MRP inhibitors and substrates including MK‐571, methotrexate, estradiol 17‐β‐d‐glucuronide, and S(2,4‐dinitrophenyl)glutathione (DNP‐SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL‐3 that inhibits high‐affinity transport of DNP‐SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low‐affinity DNP‐SG transport in human erythrocytes.
Side‐chain control of β‐peptide secondary structures
Authors: Martinek Tamás A. , Fülöp Ferenc
Published: August 2003

As one of the most important families of non‐natural polymers with the propensity to form well‐defined secondary structures, the β‐peptides are attracting increasing attention. The compounds incorporating β‐amino acid residues have found various applications in medicinal chemistry and biochemistry. The conformational pool of β‐peptides comprises several periodic folded conformations, which can be classified as helices, and nonpolar and polar strands. The latter two are prone to form pleated sheets. The numerous studies that have been performed on the side‐chain dependence of the stability of the folded structures allow certain conclusions concerning the principles of design of the β‐peptide foldamers. The folding propensity is influenced by local torsional, side‐chain to backbone and long‐range side‐chain interactions. Although β‐peptide foldamers are sensitive to solvent, the systematic choice of the side‐chain pattern and spatiality allows the design of the desired specific secondary structure. The application of β‐peptide foldamers may open up new directions in the synthesis of highly organized artificial tertiary structures with biochemical functions.
Kinetic studies on endo‐β‐galactosidase by a novel colorimetric assay and synthesis of N‐acetyllactosamine‐repeating oligosaccharide β‐glycosides using its transglycosylation activity
Authors: Murata Takeomi , Hattori Takeshi , Amarume Satoshi , Koichi Akiko , Usui Taichi
Published: August 2003

Novel chromogenic substrates for endo‐β‐galactosidase were designed on the basis of the structural features of keratan sulfate. Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (2), which consists of two repeating units of N‐acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p‐nitrophenyl β‐N‐acetyllactosaminide. In a similar manner, GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (1), GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (3), Galβ1‐4GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (4), Galβ1‐3GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (5), and Galβ1‐6GlcNAcβ1‐3Galβ1‐4Glcβ‐pNP (6) were synthesized as analogues of 2. Endo‐β‐galactosidases released GlcNAcβ‐pNP or Glcβ‐pNP in an endo‐manner from each substrate. A colorimetric assay for endo‐β‐galactosidase was developed using the synthetic substrates on the basis of the determination of p‐nitrophenol liberated from GlcNAcβ‐pNP or Glcβ‐pNP formed by the enzyme through a coupled reaction involving β‐N‐acetylhexosaminidase (β‐NAHase) or β‐d‐glucosidase. Kinetic analysis by this method showed that the value of Vmax/Km of 2 for Escherichia freundii endo‐β‐galactosidase was 1.7‐times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo‐β‐galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2‐acetamide group adjacent to the p‐nitrophenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (9) and GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ‐pNP (10) as the major products, which have N‐acetyllactosamine repeating units.
Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum)
Authors: Douglas Susan E. , Patrzykat Aleksander , Pytyck Jennifer , Gallant Jeffrey W.
Published: August 2003

Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel pleurocidin‐like antimicrobial peptides. Four genes and three pseudogenes (Ψ) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune‐related genes have been identified in upstream regions by comparative genomics. Using reverse transcription‐PCR assays, we have shown for the first time that each gene is expressed in a tissue‐specific and developmental stage‐specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides.