Organ- and species-specific biological activity of rosmarinic acid.

Rosmarinic acid (RA), a compound found in several plant species, has beneficial properties, including anti-inflammatory and antibacterial effects. We investigated the toxicity, anti-inflammatory, and antifibrotic effects of RA using precision-cut liver slices (PCLS) and precision-cut intestinal slices (PCIS) prepared from human, mouse, and rat tissue. PCLS and PCIS were cultured up to 48 h in the absence or presence of RA. Gene expression of the inflammatory markers: IL-6, IL-8/CXCL1/KC, and IL-1ß, as well as the fibrosis markers: pro-collagen 1a1, heat shock protein 47, α-smooth muscle actin, fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1) were evaluated by qPCR. RA was only toxic in murine PCIS. RA failed to mitigate the inflammatory response in most models, while it clearly reduced IL-6 and CXCL1/KC gene expression in murine PCIS at non-toxic concentrations. With regard to fibrosis, RA decreased the gene levels of Fn2 and PAI-1 in murine PCLS, and Fn2 in murine PCIS. Yet, no effect was observed on the gene expression of fibrosis markers in human and rat PCIS. In conclusion, we observed clear organ- and species-specific effects of RA. RA had little influence on inflammation. However, our study further establishes RA as a potential candidate for the treatment of liver fibrosis.

A bioinformatical and functional approach to identify novel strategies for chemoprevention of colorectal cancer.

Comparing normal colorectal mucosa and adenomas focusing on deregulated pathways obtains insight into the biological processes of early colorectal carcinogenesis. Publicly available microarray expression data from 26 normal mucosa and 47 adenoma samples were analyzed. Biological pathways enriched in adenomas were identified with Gene Set Enrichment Analysis (GSEA). The analysis revealed 10, 11 and 16 gene sets distinguishing adenomas from normal mucosa according to Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Map Annotator and Pathway Profiler (GenMAPP) and Biocarta databases, respectively. Biological pathways known to be involved in colon carcinogenesis such as cell cycle (P=0.002) and Wnt signaling (P=0.007) were enriched in adenomas. In addition, we found enrichment of novel pathways such as retinoblastoma (Rb) pathway (P=0.002), Src pathway (P=0.004), folate biosynthesis (P=0.019) and Hedgehog signaling (P=0.037) in adenomas. Microarray results for Rb and Src pathway genes were validated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on mRNA isolated from an independent set of adenoma and normal colon samples. A high correlation between microarray data and qRT-PCR expression data was found. The relevance of targeting of identified pathways was shown using the Rb pathway inhibitors roscovitine and PD-0332991 and the Src pathway inhibitor dasatinib. All inhibitors used induced cell growth reduction in adenoma cells. This study shows a bioinformatical and functional approach leading to potentially new options for chemoprevention of colorectal cancer.

An ex vivo human model system to evaluate specificity of replicating and non-replicating gene therapy agents.

BACKGROUND: Inefficiency, aspecificity and toxicity of gene transfer vectors hamper gene therapy from showing its full potential. On this basis significant research currently focuses on developing vectors with improved infection and/or expression profiles. Screening assays with validity to the clinical context to determine improved characteristics of such agents are not readily available since this requires a close relationship to the human situation. We present a clinically relevant tissue slice technology to preclinically test improved vector characteristics. METHODS: Slices were prepared from rat, mouse and human liver samples and from tumor tissue. Specificity of gene expression and replication was determined by infecting target and non-target tissue slices with transcriptionally retargeted adenoviruses and oncolytic viruses. RESULTS: Using rat liver slices, we demonstrate efficient knob-mediated adenoviral infectivity. A favorable tumor-on/liver-off profile, resembling in vitro and mouse in vivo data, was shown for a tumor-specific transcriptionally retargeted adenovirus by infecting slices prepared from tumor or liver tissue. Similar liver-off data were found for mouse, rat and human samples (over 3-log lower activity of the tumor-specific promoter compared to cytomegalovirus (CMV)). More importantly, we show that this technology when applied to human livers is a powerful tool to determine aspecific replication of oncolytic viruses in liver tissue. A 2- to 6-log reduction in viral replication was observed for a tumor-specific oncolytic virus compared to the wild-type adenovirus. CONCLUSIONS: The precision-cut tissue slice technology is a powerful method to test specificity and efficiency of gene transfer as well as of viral replication using human tissue.

Influence of Meloidogyne incognita on the Water Relations of Cotton Grown in Microplots.

The effects of Meloidogyne incognita on the growth and water relations of cotton were evaluated in a 2-year field study. Microplots containing methyl bromide-fumigated fine sandy loam soil were infested with the nematode and planted to cotton (Gossypium hirsutum L.). Treatments included addition of nematodes alone, addition of nematodes plus the insecticide-nematicide aldicarb (1.7 kg/ha), and an untreated control. Meloidogyne incognita population densities reached high levels in both treatments where nematodes were included. Root galling, plant height at harvest, and seed cotton yield were decreased by nematode infection. In older plants (89 days after planting [DAP]), leaf transpiration rates and stomatal conductance were reduced, and leaf temperature was increased by nematode infection. Nematode infection did not affect (P = 0.05) leaf water potential in either young or older plants but lowered the osmotic potential. The maximum rate and cumulative amount of water flowing through intact plants during a 24-hour period were lower, on both a whole-plant and per-unit-leaf-area basis, in infected plants than in control plants. Application of aldicarb moderated some of the nematode effects but did not eliminate them.

Evidence for light-dependent recycling of respired carbon dioxide by the cotton fruit.

Conservation of respired CO(2) by an efficient recycling mechanism in fruit could provide a significant source of C for yield productivity. However, the extent to which such a mechanism operates in cotton (Gossypium hirsutum L.) is unknown. Therefore, a combination of CO(2) exchange, stable C isotope, and chlorophyll (Chl) fluorescence techniques were used to examine the recycling of respired CO(2) in cotton fruit. Respiratory CO(2) losses of illuminated fruit were reduced 15 to 20% compared with losses for dark-incubated fruit. This light-dependent reduction in CO(2) efflux occurred almost exclusively via the fruit's outer capsule wall. Compared with the photosynthetic activity of leaves, CO(2) recycling by the outer capsule wall was 35 to 40% as efficient. Calculation of (14)CO(2) fixation on a per Chl basis revealed that the rate of CO(2) recycling for the capsule wall was 62.2 micromoles (14)CO(2) per millimole Chl per second compared with an assimilation rate of 64.6 micromoles (14)CO(2) per millimole Chl per second for leaves. During fruit development, CO(2) recycling contributed more than 10% of that C necessary for fruit dry weight growth. Carbon isotope analyses (delta(13)C) showed significant differences among the organs examined, but the observed isotopic compositions were consistent with a C(3) pathway of photosynthesis. Pulse-modulated Chl fluorescence indicated that leaves and fruit were equally efficient in photochemical and nonphotochemical dissipation of light energy. These studies demonstrated that the cotton fruit possesses a highly efficient, light-dependent CO(2) recovery mechanism that aids in the net retention of plant C and, therein, contributes to yield productivity.

Interaction of Meloidogyne incognita and Water Stress in Two Cotton Cultivars.

A series of controlled-environment experiments were conducted to elucidate the effects of Meloidogyne incognita on host physiology and plant-water relations of two cotton (Gossypium hirsutum) cultivars that differed in their susceptibility to nematode infection. Inoculation of M. incognita-resistant cultivar Auburn 634 did not affect growth, stomatal resistance, or components of plant-water potential relative to uninoculated controls. However, nematode infection of the susceptible cultivar Stoneville 506 greatly suppressed water flow through intact roots. This inhibition exceeded 28% on a root-length basis and was similar to that observed as a consequence of severe water stress in a high evaporative demand environment. Nematodes did not affect the components of leaf water potential, stomatal resistance, transpiration, or leaf temperature. However, these factors were affected by the interaction of M. incognita and water stress. Our results indicate that M. incognita infection may alter host-plant water balance and may be a significant factor in early-season stress on cotton seedlings.

Photosynthetic and Respiratory Activity of Fruiting Forms within the Cotton Canopy.

The supply of photosynthates by leaves for reproductive development in cotton (Gossypium hirsutum L.) has been extensively studied. However, the contribution of assimilates derived from the fruiting forms themselves is inconclusive. Field experiments were conducted to document the photosynthetic and respiratory activity of cotton leaves, bracts, and capsule walls from anthesis to fruit maturity. Bracts achieved peak photosynthetic rates of 2.1 micromoles per square meter per second compared with 16.5 micromoles per square meter per second for the subtending leaf. However, unlike the subtending leaf, the bracts did not show a dramatic decline in photosynthesis with increased age, nor was their photosynthesis as sensitive as leaves to low light and water-deficit stress. The capsule wall was only a minor site of (14)CO(2) fixation from the ambient atmosphere. Dark respiration by the developing fruit averaged -18.7 micromoles per square meter per second for 6 days after anthesis and declined to -2.7 micromoles per square meter per second after 40 days. Respiratory loss of CO(2) was maximal at -158 micromoles CO(2) per fruit per hour at 20 days anthesis. Diurnal patterns of dark respiration for the fruit were age dependent and closely correlated with stomatal conductance of the capsule wall. Stomata on the capsule wall of young fruit were functional, but lost this capacity with increasing age. Labeled (14)CO(2) injected into the fruit interior was rapidly assimilated by the capsule wall in the light but not in the dark, while fiber and seed together fixed significant amounts of (14)CO(2) in both the light and dark. These data suggest that cotton fruiting forms, although sites of significant respiratory CO(2) loss, do serve a vital role in the recycling of internal CO(2) and therein, function as important sources of assimilate for reproductive development.

Photosynthesis of individual field-grown cotton leaves during ontogeny.

Photosynthetic characteristics of field-grown cotton (Gossypium hirsutum L.) leaves were determined at several insertion levels within the canopy during the growing season. Single-leaf measurements of net photosynthesis (Pn), stomatal conductance to CO2 (gs·CO2), substomatal CO2, leaf area expansion, leaf nitrogen, and light intensity (PPFD) were recorded for undisturbed leaves within the crop canopy at 3-4 day intervals during the development of all leaves at main-stem nodes 8, 10, and 12. Patterns of Pn during leaf ontogeny exhibited three distinct phases; a rapid increase to maximum at 16-20 days after leaf unfolding, a relatively short plateau, and a period of linear decline to negligible Pn at 60-65 days. Analysis of the parameters which contributed to the rise and fall pattern of Pn with leaf age indicated the primary involvement of leaf area expansion, leaf nitrogen, PPFD, and gs·CO2 in this process. The response of Pn and gs·CO2 to incident PPFD conditions during canopy development was highly age dependent. For leaves less than 16 days old, the patterns of Pn and gs·CO2 were largely controlled by non-PPFD factors, while for older leaves Pn and gs·CO2 were more closely coupled to PPFD-mediated processes. Maximum values of Pn were not significantly different for any of the leaves monitored in this study, however, those leaves at main-stem node 8 did possess a significantly diminished photosynthetic capacity with age compared to upper canopy leaves. This accelerated decline in Pn could not be explained by age-related variations in gs·CO2 since all leaves showed similar changes in gs·CO2 with leaf age.

Osmotic Adjustment in Cotton (Gossypium hirsutum L.) Leaves and Roots in Response to Water Stress.

The relative magnitude of adjustment in osmotic potential (psi(s)) of water-stressed cotton (Gossypium hirsutum L.) leaves and roots was studied using plants raised in pots of sand and grown in a growth chamber. One and three water-stress preconditioning cycles were imposed by withholding water, and the subsequent adjustment in solute potential upon relief of the stress and complete rehydration was monitored with thermocouple psychrometers. Both leaves and roots exhibited a substantial adjustment in psi(s) in response to water stress with the former exhibiting the larger absolute adjustment. The osmotic adjustment of leaves was 0.41 megapascal compared to 0.19 megapascal in the roots. The roots, however, exhibited much larger percentage osmotic adjustments of 46 and 63% in the one and three stress cycles, respectively, compared to 22 and 40% in the leaves in similar stress cycles. The osmotically adjusted condition of leaves and roots decreased after relief of the single cycle stress to about half the initial value within 3 days, and to the well-watered control level within 6 days. In contrast, increasing the number of water-stress preconditioning cycles resulted in significant percentage osmotic adjustment still being present after 6 days in roots but not in the leaves. The decrease in psi(s) of leaves persisted longer in field-grown cotton plants compared to plants of the same age grown in the growth chamber. The advantage of decreased psi(s) in leaves and roots of water-stressed cotton plants was associated with the maintenance of turgor during periods of decreasing water potentials.

A rapid leaf-disc sampler for psychrometric water potential measurements.

An instrument was designed which facilitates faster and more accurate sampling of leaf discs for psychrometric water potential measurements. The instrument consists of an aluminum housing, a spring-loaded plunger, and a modified brass-plated cork borer. The leaf-disc sampler was compared with the conventional method of sampling discs for measurement of leaf water potential with thermocouple psychrometers on a range of plant material including Gossypium hirsutum L., Zea mays L., and Begonia rex-cultorum L. The new sampler permitted a leaf disc to be excised and inserted into the psychrometer sample chamber in less than 7 seconds, which was more than twice as fast as the conventional method. This resulted in more accurate determinations of leaf water potential due to reduced evaporative water losses. The leaf-disc sampler also significantly reduced sample variability between individual measurements. This instrument can be used for many other laboratory and field measurements that necessitate leaf disc sampling.

Ratio of cut surface area to leaf sample volume for water potential measurements by thermocouple psychrometers.

Evaporative losses from the cut edge of leaf samples are of considerable importance in measurements of leaf water potential using thermocouple psychrometers. The ratio of cut surface area to leaf sample volume (area to volume ratio) has been used to give an estimate of possible effects of evaporative loss in relation to sample size. A wide range of sample sizes with different area to volume ratios has been used. Our results using Glycine max L. Merr. cv Bragg indicate that leaf samples with area to volume values less than 0.2 square millimeter per cubic millimeter give psychrometric leaf water potential measurements that compare favorably with pressure chamber measurements.