Does usnic acid affect microtubules in human cancer cells? / O ácido úsnico pode afetar microtúbulos em células cancerosas humanas?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.

Does usnic acid affect microtubules in human cancer cells?

Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 microM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.

Interprofessional attitudes and perceptions: Results from a longitudinal controlled trial of pre-registration health and social care students in Scotland.

This study made use of a controlled longitudinal design to assess the impact on pre-registration health and social care students of an interprofessional intervention on the attitudes to and perceptions of interprofessional ideals. Evaluation, over four years, of Nursing, Occupational Therapy, Podiatry, Prosthetics and Orthotics, Physiotherapy and Radiography students was performed using the adapted versions of the Readiness for Interprofessional Learning Scale (RIPLS) and the Interdisciplinary Education Perception Scale (IEPS). Baseline samples of the control and experimental groups were 260 and 313 respectively. Support for Interprofessional Education (IPE) appears high but possibly idealistically so initially. Restricted Maximum Likelihood (REML) models were used to assess intervention effects as well as any possible profession or time effects. The intervention was found to have had a significant effect on five of the measured sub-scales and the professions were found to react in a significantly different way on four of the sub-scales. The inclusion of a control group has confirmed previous findings from other studies but also highlights the possible effects of the general learning and teaching methodologies employed within various professions as well as the need for research into the influence of the timing, duration, style and content of clinical placement periods.

Prevalence of vitamin deficiencies on admission: relationship to hospital mortality in critically ill patients.

Vitamin deficiency is believed to be common in critical illness. Water soluble and antioxidant vitamins are those most frequently used for supplementation in these patients. There are no data to confirm the prevalence of vitamin deficiencies in high-risk emergently admitted intensive care patients, nor their association with hospital mortality. One hundred and twenty-nine consecutive, critically ill patients who were emergently admitted to intensive care were enrolled in this prospective observational cohort study. Patient data including diagnosis, source of admission and severity of illness scores were prospectively collected. Within the first 48 hours of admission, concentrations of C-reactive protein, Vitamins A, E, B1, B12 and folate were measured on arterial blood. Multivariate stepwise logistic regression modelling was performed to examine the association of vitamin concentrations with hospital mortality. Fifty-five patients (43%) had a biochemical deficiency of one of the five vitamins on admission to the intensive care unit. A total of 18 patients died (14%) during their hospital stay (15 of those in the intensive care unit). Moderate correlations with C-reactive protein concentrations were demonstrated for Vitamins B12, A and E (Spearman's r = 0.309, -0.541 and -0.299, P = 0.001, 0.001 and 0.007 respectively). Hospital mortality was significantly associated with age, APACHE II score, admission and maximum Sequential Organ Failure Assessment scores and admission source in the univariate analyses. Multivariate analysis did not demonstrate an association between biochemical deficiency and mortality. Biochemical deficiencies of water-soluble and antioxidant vitamins are common on admission in unplanned or emergency admissions to the intensive care unit, but we could not demonstrate an independent association with hospital mortality.

A calorimetric study of dimyristoylphosphatidylcholine phase transitions and steroid-liposome interactions for liposomes prepared by thin film and proliposome methods.

Using high sensitivity differential scanning calorimetry (HSDSC), the phase transitions of dimyristoylphosphatidylcholine (DMPC) liposomal bilayers and their interaction with the model steroid beclomethasone dipropionate (BDP) were found to be dependent on the method of liposome manufacture. Ethanol-based proliposomes produced liposomes having no phospholipid pretransition, a main transition of high enthalpy and a low onset temperature, and a very low incorporation of the steroid (maximum 1 mol%). This was attributed to an alcohol-induced interdigitation of the bilayers, which was not apparently reversed by flushing the liposome dispersion with nitrogen in an attempt to remove ethanol. For liposomes manufactured by thin film or particulate-based proliposome methods, 1-2.5 mol% steroid was optimal for incorporation within bilayers, although the nature of the steroid interaction with the bilayers differed between the two methods. For liposomes manufactured by the thin film method, a higher steroid concentration resulted in a broadened main transition and a reduced melting cooperativity. This suggests that BDP formed separate domains within the bilayers which caused non-ideal mixing and phase separation at 5 mol% steroid. This observation was absent for liposomes generated from particulate-based proliposomes, indicating separate steroid domains were not formed and subsequent non-ideal mixing and phase separation did not occur. In addition, liposomes generated from particulate-based proliposomes showed reduced pretransition and main transition enthalpies. These differences were attributed to the employment of sucrose to manufacture the particulate-based proliposomes. This study has shown that the thermal behaviour of liposomes and their interaction with beclomethasone dipropionate were dependent on the method of liposome manufacture. Moreover, particulate-based proliposomes may provide a reasonable alternative to the conventional thin film method in producing liposomes incorporating this steroid.

Recent developments for the analysis of data obtained from isothermal calorimetry.

Isothermal calorimetry is rapidly becoming an indispensable tool for the quantitative determination of a variety of kinetic and thermodynamic parameters for a wide range of systems. In particular calorimetry is finding increased application to the investigation of stability and incompatibility of pharmaceutical materials. In order to draw meaningful conclusions and to predict behaviour in related systems it is necessary to have the means to calculate accurately parameters such as the rate constant and enthalpy. To this end several groups have been developing equations which describe calorimetric output in these terms. This paper will briefly outline some of these equations and discuss some of the limitations that currently exist in their application. A particular emphasis is placed on the recent developments relating to the application of these equations to flow calorimetric data. The main application of these equations is usually found in the pharmaceutical industry. Pharmaceutical formulations are usually extremely complex mixtures consisting of many different excipients as well as the active drug. Because of these large numbers of ingredients it is often observed that multiple chemical and physical process occur over the lifetime of the study. This complexity is then reflected in the calorimetric data rendering the application of the simple equations useless. Dealing with this complexity is a major issue amongst the calorimetric community and some of the recent advances in this field are also discussed.

Doxorubicin and vinorelbine act independently via p53 expression and p38 activation respectively in breast cancer cell lines.

In the treatment of breast cancer, combination chemotherapy is used to overcome drug resistance. Combining doxorubicin and vinorelbine in the treatment of patients with metastatic breast cancer has shown high response rates; even single-agent vinorelbine in patients previously exposed to anthracyclines results in significant remission. Alterations in protein kinase-mediated signal transduction and p53 mutations may play a role in drug resistance with cross-talk between signal transduction and p53 pathways. The aim of this study was to establish the effects of doxorubicin and vinorelbine, as single agents, in combination, and as sequential treatments, on signal transduction and p53 in the breast cancer cell lines MCF-7 and MDA-MB-468. In both cell lines, increased p38 activity was demonstrated following vinorelbine but not doxorubicin treatment, whether vinorelbine was given prior to or simultaneously with doxorubicin. Mitogen-activated protein kinase (MAPK) activity and p53 expression remained unchanged following vinorelbine treatment. Doxorubicin treatment resulted in increased p53 expression, without changes in MAPK or p38 activity. These findings suggest that the effect of doxorubicin and vinorelbine used in combination may be achieved at least in part through distinct mechanisms. This additivism, where doxorubicin acts via p53 expression and vinorelbine through p38 activation, may contribute to the high clinical response rate when the two drugs are used together in the treatment of breast cancer.

Characterization of a family of Arabidopsis genes related to xyloglucan fucosyltransferase1.

To understand primary cell wall assembly in Arabidopsis, we have focused on identifying and characterizing enzymes involved in xyloglucan biosynthesis. Nine genes (AtFUT2-10) were identified that share between 47% and 62% amino acid similarity with the xyloglucan-specific fucosyltransferase AtFUT1. Reverse transcriptase-PCR analysis indicates that all these genes are expressed. Bioinformatic analysis predicts that these family members are fucosyltransferases, and we first hypothesized that some may also be involved in xyloglucan biosynthesis. AtFUT3, AtFUT4, and AtFUT5 were expressed in tobacco (Nicotiana tabacum L. cv BY2) suspension culture cells, and the resulting proteins did not transfer fucose (Fuc) from GDP-Fuc to tamarind xyloglucan. AtFUT3, AtFUT4, and AtFUT5 were overexpressed in Arabidopsis plants. Leaves of plants overexpressing AtFUT4 or AtFUT5 contained more Fuc than wild-type plants. Stems of plants overexpressing AtFUT4 or AtFUT5 contained more xylose, less arabinose, and less galactose than wild-type plants. We suggest that the AtFUT family is likely to include fucosyltransferases important for the synthesis of wall carbohydrates. A targeted analysis of isolated cell wall matrix components from plants altered in expression of these proteins will help determine their specificity and biological function.

Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth.

Turgor-driven plant cell growth depends on wall structure. Two allelic l-fucose-deficient Arabidopsis thaliana mutants (mur1-1 and 1-2) are dwarfed and their rosette leaves do not grow normally. mur1 leaf cell walls contain normal amounts of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II), but only half exists as a borate cross-linked dimer. The altered structure of mur1 RG-II reduces the rate of formation and stability of this cross-link. Exogenous aqueous borate rescues the defect. The reduced cross-linking of RG-II in dwarf mur1 plants indicates that plant growth depends on wall pectic polysaccharide organization.

The portable UNIX programming system (PUPS) and CANTOR: a computational environment for dynamical representation and analysis of complex neurobiological data.

Many problems in analytical biology, such as the classification of organisms, the modelling of macromolecules, or the structural analysis of metabolic or neural networks, involve complex relational data. Here, we describe a software environment, the portable UNIX programming system (PUPS), which has been developed to allow efficient computational representation and analysis of such data. The system can also be used as a general development tool for database and classification applications. As the complexity of analytical biology problems may lead to computation times of several days or weeks even on powerful computer hardware, the PUPS environment gives support for persistent computations by providing mechanisms for dynamic interaction and homeostatic protection of processes. Biological objects and their interrelations are also represented in a homeostatic way in PUPS. Object relationships are maintained and updated by the objects themselves, thus providing a flexible, scalable and current data representation. Based on the PUPS environment, we have developed an optimization package, CANTOR, which can be applied to a wide range of relational data and which has been employed in different analyses of neuroanatomical connectivity. The CANTOR package makes use of the PUPS system features by modifying candidate arrangements of objects within the system's database. This restructuring is carried out via optimization algorithms that are based on user-defined cost functions, thus providing flexible and powerful tools for the structural analysis of the database content. The use of stochastic optimization also enables the CANTOR system to deal effectively with incomplete and inconsistent data. Prototypical forms of PUPS and CANTOR have been coded and used successfully in the analysis of anatomical and functional mammalian brain connectivity, involving complex and inconsistent experimental data. In addition, PUPS has been used for solving multivariate engineering optimization problems and to implement the digital identification system (DAISY), a system for the automated classification of biological objects. PUPS is implemented in ANSI-C under the POSIX.1 standard and is to a great extent architecture- and operating-system independent. The software is supported by systems libraries that allow multi-threading (the concurrent processing of several database operations), as well as the distribution of the dynamic data objects and library operations over clusters of computers. These attributes make the system easily scalable, and in principle allow the representation and analysis of arbitrarily large sets of relational data. PUPS and CANTOR are freely distributed (http://www.pups.org.uk) as open-source software under the GNU license agreement.

Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.

Pectin is a family of complex polysaccharides present in all plant primary cell walls. The complicated structure of the pectic polysaccharides, and the retention by plants of the large number of genes required to synthesize pectin, suggests that pectins have multiple functions in plant growth and development. In this review we summarize the current level of understanding of pectin primary and tertiary structure, and describe new methods that may be useful to study localized pectin structure in the plant cell wall. We also discuss progress in our understanding of how pectin is biosynthesized and review the biological activities and possible modes of action of pectic oligosaccharides referred to as oligogalacturonides. We present our view of critical questions regarding pectin structure, biosynthesis, and function that need to be addressed in the coming decade. As the plant community works towards understanding the functions of the tens of thousands of genes expressed by plants, a large number of those genes are likely to be involved in the synthesis, turnover, biological activity, and restructuring of pectin. A combination of genetic, molecular, biochemical and chemical approaches will be necessary to fully understand the function and biosynthesis of pectin.

Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain.

Monomeric rhamnogalacturonan II (mRG-II) was isolated from red wine and the reducing-end galacturonic acid of the backbone converted to L-galactonic acid by treatment with NaBH4. The resulting product (mRG-II'ol) was treated with a cell-free extract from Penicillium daleae, a fungus that has been shown to produce RG-II-fragmenting glycanases. The enzymatically generated products were fractionated by size-exclusion and anion-exchange chromatographies and the quantitatively major oligosaccharide fraction isolated. This fraction contained structurally related oligosaccharides that differed only in the presence or absence of a single Kdo residue. The Kdo residue was removed by acid hydrolysis and the resulting oligosaccharide then characterized by 1- and 2D 1H NMR spectroscopy, ESMS, and by glycosyl-residue and glycosyl-linkage composition analyses. The results of these analyses provide evidence for the presence of at least two structurally related oligosaccharides in the ratio approximately 6:1. The backbone of these oligosaccharides is composed of five (1-->4)-linked alpha-D-GalpA residues and a (1-->3)-linked L-galactonate. The (1-->4)-linked GalpA residue adjacent to the terminal non-reducing GalpA residue of the backbone is substituted at O-2 with an apiosyl-containing side chain. Beta3-L-Araf-(1-->5)-beta-D-DhapA is likely to be linked to O-3 of the GalpA residue at the non-reducing end of the backbone in the quantitatively major oligosaccharide and to O-3 of a (1-->4)-linked GalpA residue in the backbone of the minor oligosaccharide. Furthermore, the results of our studies have shown that the enzymically generated aceryl acid-containing oligosaccharide contains an alpha-linked aceryl acid residue and a beta-linked galactosyl residue. Thus, the anomeric linkages of these residues in RG-II should be revised.

Anatomical connectivity defines the organization of clusters of cortical areas in the macaque monkey and the cat.

The number of different cortical structures in mammalian brains and the number of extrinsic fibres linking these regions are both large. As with any complex system, systematic analysis is required to draw reliable conclusions about the organization of the complex neural networks comprising these numerous elements. One aspect of organization that has long been suspected is that cortical networks are organized into 'streams' or 'systems'. Here we report computational analyses capable of showing whether clusters of strongly interconnected areas are aspects of the global organization of cortical systems in macaque and cat. We used two different approaches to analyse compilations of corticocortical connection data from the macaque and the cat. The first approach, optimal set analysis, employed an explicit definition of a neural 'system' or 'stream', which was based on differential connectivity. We defined a two-component cost function that described the cost of the global cluster arrangement of areas in terms of the areas' connectivity within and between candidate clusters. Optimal cluster arrangements of cortical areas were then selected computationally from the very many possible arrangements, using an evolutionary optimization algorithm. The second approach, non-parametric cluster analysis (NPCA), grouped cortical areas on the basis of their proximity in multidimensional scaling representations. We used non-metric multidimensional scaling to represent the cortical connectivity structures metrically in two and five dimensions. NPCA then analysed these representations to determine the nature of the clusters for a wide range of different cluster shape parameters. The results from both approaches largely agreed. They showed that macaque and cat cortices are organized into densely intra-connected clusters of areas, and identified the constituent members of the clusters. These clusters reflected functionally specialized sets of cortical areas, suggesting that structure and function are closely linked at this gross, systems level.

Hierarchical organization of macaque and cat cortical sensory systems explored with a novel network processor.

Neuroanatomists have described a large number of connections between the various structures of monkey and cat cortical sensory systems. Because of the complexity of the connection data, analysis is required to unravel what principles of organization they imply. To date, analysis of laminar origin and termination connection data to reveal hierarchical relationships between the cortical areas has been the most widely acknowledged approach. We programmed a network processor that searches for optimal hierarchical orderings of cortical areas given known hierarchical constraints and rules for their interpretation. For all cortical systems and all cost functions, the processor found a multitude of equally low-cost hierarchies. Laminar hierarchical constraints that are presently available in the anatomical literature were therefore insufficient to constrain a unique ordering for any of the sensory systems we analysed. Hierarchical orderings of the monkey visual system that have been widely reported, but which were derived by hand, were not among the optimal orderings. All the cortical systems we studied displayed a significant degree of hierarchical organization, and the anatomical constraints from the monkey visual and somato-motor systems were satisfied with very few constraint violations in the optimal hierarchies. The visual and somato-motor systems in that animal were therefore surprisingly strictly hierarchical. Most inconsistencies between the constraints and the hierarchical relationships in the optimal structures for the visual system were related to connections of area FST (fundus of superior temporal sulcus). We found that the hierarchical solutions could be further improved by assuming that FST consists of two areas, which differ in the nature of their projections. Indeed, we found that perfect hierarchical arrangements of the primate visual system, without any violation of anatomical constraints, could be obtained under two reasonable conditions, namely the subdivision of FST into two distinct areas, whose connectivity we predict, and the abolition of at least one of the less reliable rule constraints. Our analyses showed that the future collection of the same type of laminar constraints, or the inclusion of new hierarchical constraints from thalamocortical connections, will not resolve the problem of multiple optimal hierarchical representations for the primate visual system. Further data, however, may help to specify the relative ordering of some more areas. This indeterminacy of the visual hierarchy is in part due to the reported absence of some connections between cortical areas. These absences are consistent with limited cross-talk between differentiated processing streams in the system. Hence, hierarchical representation of the visual system is affected by, and must take into account, other organizational features, such as processing streams.

Computational analysis of functional connectivity between areas of primate cerebral cortex.

Recent analyses of association fibre networks in the primate cerebral cortex have revealed a small number of densely intra-connected and hierarchically organized structural systems. Corresponding analyses of data on functional connectivity are required to establish the significance of these structural systems. We therefore built up a relational database by systematically collating published data on the spread of activity after strychnine-induced disinhibition in the macaque cerebral cortex in vivo. After mapping these data to two different parcellation schemes, we used three independent methods of analysis which demonstrate that the cortical network of functional interactions is not homogeneous, but shows a clear segregation into functional assemblies of mutually interacting areas. The assemblies suggest a principal division of the cortex into visual, somatomotor and orbito-temporo-insular systems, while motor and somatosensory areas are inseparably interrelated. These results are largely compatible with corresponding analyses of structural data of mammalian cerebral cortex, and deliver the first functional evidence for 'small-world' architecture of primate cerebral cortex.

Ultrastructural aspects of the microvasculature of the cochlea: the internal spiral network.

We performed a study of the microvasculature of the cochlea with scanning electron microscopy on corrosion casts in the guinea pig. This study was performed in 140 cochleas from healthy adult guinea pigs. Different microvascular techniques were used, including injection-microdissection in 53 cases, injection-diaphanization in 27 cases, a histologic technique in 34 cases, injection-microdissection-diaphanization in 32 cases, and scanning electron microscopy on corrosion casts in 14 cases. The internal radiating arterioles branch off as collateral branches of the spiral modiolar artery, as first-order collateral branches. We detail the morphology, caliber, trajectory, and collateral branches. Among their collateral branches, are third-order arterioles, the arterioles to the spiral ganglia, and the arterioles of the tympanic lip. The arterioles of the tympanic lip form, through their anastomosis, a rich capillary network at the edge of the spiral lamina, called the internal spiral network. We emphasize the glomeruli of Schwalbe, which arise near the scala tympani as third-order arterioles with a medium caliber of 14 microm. The upper glomeruli, situated in the bony wall, and the lower glomeruli, situated in the width of the osseous spiral lamina, form vascular loops made of anastomosed capillaries with a caliber of less than 10 microm. These loops play an important role as efficient devices, or "relay stations," for regulation of cochlear blood flow. The comparison of results obtained with each technique gave the perspective of the cochlear microvasculature with great accuracy.

The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer.

The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-O-methyl-D-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and in in vitro synthesized dimer. Pb2+, La3+, and Ca2+ increase dimer formation in vitro in a concentration- and pH-dependent manner. Pb2+ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mM) is treated for 5 min at pH 3.5 with boric acid (1 mM) and Pb2+ (0.5 mM); at pH 5 the rate of conversion is somewhat slower. Hg2+ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers self-assemble to form a dimer.

Early-onset inflammatory responses in vivo to adenoviral vectors in the presence or absence of lipopolysaccharide-induced inflammation.

Adenoviral vectors (Ad) have potential for use in pulmonary gene transfer for treating cystic fibrosis (CF). However, Ad may induce inflammation even in the absence of gene expression. Endotoxin from gram-negative bacteria in the airways of CF patients may also induce inflammation, and may further inhibit vector delivery and gene transfer. We used a mouse model to study the time course of Ad-induced lung inflammation and to assess additivity with lipopolysaccharide (LPS)-induced inflammatory responses. C3H/HeJ endotoxin-resistant (RES) mice hyporesponsive to inflammatory stimuli and normoresponsive C3HeB/FeJ endotoxin-sensitive (SEN) mice were studied to characterize inflammatory responses that follow intratracheal instillation of inactivated Ad, with or without simultaneous inhalation exposure to LPS. Instillation of 10(10) Ad particles dramatically increased bronchoalveolar lavage fluid (BALF) concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 at 3 to 6 h and induced profound neutrophilia, maximal at 12 to 24 h. SEN mice had tenfold greater responses than did RES mice at 6, 12, and 24 h. Mice exposed to Ad alone, LPS alone, or Ad + LPS had significant inflammation at the 3-h time point as demonstrated by BALF neutrophils, TNF-alpha, and IL-6. With all three treatments, SEN mice had a five- to 300-fold greater response than did RES mice. Importantly, Ad + LPS yielded no greater inflammatory response than LPS without Ad. These data demonstrate that replication-deficient Ad induce early inflammation and LPS-induced inflammation is not augmented by concurrent treatment with Ad.

Biological activity of reducing-end-derivatized oligogalacturonides in tobacco tissue cultures.

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H2O2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.