Stable carbon and nitrogen isotope signatures of decomposing tropical macrophytes

The Pantanal of Mato Grosso, Brazil, is a large, seasonal wetland, which exhibits high macrophyte productivity at the beginning of the rainy season, when the floodplain becomes flooded. During inundation, from December through May, there is rapid turnover of decomposing macrophyte litter, which is subsequently colonized and consumed by various organisms. In this paper, the variation in the carbon and nitrogen isotope signatures of decomposing macrophytes and detritus was determined to provide an isotopic baseline for the elucidation of higher trophic levels. Seven abundant macrophyte species, Cyperaceae sp., Pontederia lanceolata, Cabomba furcata, Salvinia auriculata, Eichhornia crassipes, Nymphaea amazonum and Paspalum repens, were exposed in mesocosm decomposition experiments lasting 21 or 100 days. Stable isotope ratios of carbon and nitrogen and the atomic C/N ratios were determined for decomposing plant material, particulate organic matter (POM), the microbial film, and aquatic invertebrate larvae. The ¹³C values for the macrophytes did not change during decomposition. However, the variability of ¹⁵N was high (range of ± 6 ) due to microbial activity. There was no consistent difference in the isotopic signatures of macrophytes and POM. C/N ratios decreased from 17 to 50 in macrophytes, to 7 to 12 in POM. The isotopic signatures and C/N ratios of the microbial film were the same as those of POM. We concluded that heterotrophic processes did not fractionate stable carbon isotopes but caused an increase in the variability of stable nitrogen ratios and a change in the C/N ratios in our experimental system. Therefore, it was not possible to distinguish fresh and senescent material or even POM when used as a food source. The ¹³C values of the aquatic larvae were closely coupled to those of the carbon source provided.     

Mactinin: a modulator of the monocyte response to inflammation

During inflammatory processes, monocytes leave the blood stream at increased rates and enter inflammation tissue, where they undergo phenotypic transformation to mature macrophages with enhanced phagocytic activity. alpha-Actinin, a cytoskeletal protein, is present in focal adhesion complexes and left in the microenvironment as a result of cell movement. Mactinin, a 31 kDa amino-terminal fragment of alpha-actinin, is generated by the degradation of extracellular alpha-actinin by monocyte-secreted urokinase. We have previously demonstrated that mactinin promotes monocyte/macrophage maturation. We now report that 0.5-10 nM mactinin has significant chemotactic activity for monocytes. Mactinin seems to be present in inflammatory arthritis synovial fluid, because affinity-purified antisera reacted with a protein of the expected molecular mass in various types of arthritis fluids that were immunoaffinity-purified and subjected to Western analysis. Thus, six of seven samples from patients with psoriatic arthritis, reactive arthritis, gout, or ankylosing spondylitis contained mactinin at levels that are active in vitro. Initially, mactinin was not found in affinity-purified rheumatoid arthritis samples. However, it was detectable after the dissociation of immune complexes, suggesting that it was complexed to anti-microfilament auto-antibodies. In addition, mactinin was found in the lavage fluid from the arthritic knee joints of rabbits with antigen-induced arthritis and was absent from the contralateral control knee fluids. We conclude that mactinin is present in several types of inflammatory arthritis and might modulate mononuclear phagocyte response to inflammation.     

Characterization of Ricinus communis phloem profilin,RcPRO1

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.     

Meningeal cells stimulate and direct the migration of cerebellar external granule cells in vitro

The external granular layer is a secondary proliferative zone that arises from the caudolateral margin of the cerebellar ventricular zone and then spreads beneath the pial surface, eventually covering the entire cerebellar anlage. Here, both a part of the Bergmann glia and granule cells are generated. Selective destruction of the leptomeningeal cell layer during development in vivo disrupts the subpial extension of the external granular layer and the laminar deposition of its descendant cells. The mechanisms by which meningeal fibroblasts exert their controlling influence on cortical development have remained unclear but could involve diffusible factors and/or interactions mediated by direct cellular contacts. In order to test these assumptions, we have co-cultivated cerebellar slice explants with meningeal cells with and without interposition of a microfilter barrier. In this setup, meningeal cells by a diffusible factor stimulated the emigration of immature neurons exclusively from the external granular layer. This effect could also be elicited by fibroblasts from other tissues but not by nonfibroblastic cells such as, e.g., astroglia. In the Boyden chamber assay, the migration of undifferentiated neurons isolated from the external granular layer was chemotactically oriented towards the source of meningeal cell-conditioned media. In comparison, neurons from the internal granular layer did not respond to this stimulus. The attraction of immature neurons towards the pial surface could (1) represent a mechanism for the establishment of (subpial) secondary proliferative zones and (2) hypothetically also play a role in the outward-directed migration of postmitotic cells, e.g., in the isocortical anlage.     

Hybridisation between oilseed rape (Brassica napus) and tetraploid Brassica rapa under field conditions

Cross-compatible relatives of crop species contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified varieties. The most successful crossing partner of oilseed rape (Brassica napus L.) is diploid Brassica rapa L. Variation of ploidy level among B. rapa cultivars has, until recently, been neglected in the context of gene flow and hybridisation with oilseed rape. We estimated the extent of hybridisation between autotetraploid B. rapa varieties (female) and B. napus (pollen donor) under experimental field conditions. Morphology, variation of relative DNA amount, and microsatellite markers were used to distinguish between intraspecific offspring of tetraploid B. rapa and interspecific hybrids with B. napus. Of 517 seed progenies of tetraploid B. rapa, 45 juvenile plants showed species specific morphological traits of oilseed rape. The detection of putative hybrids based on variation in relative DNA amounts was problematic due to the occurrence of aneuploidy. In total, 84 offspring showed relative DNA amounts deviating from tetraploid B. rapa, four of which were hexaploids. Of the 205 offspring analysed at three microsatellite loci, 67 had oilseed rape alleles. Based on molecular evidence a minimum hybridisation rate of 13.0% was estimated. A few mother plants accounted for the majority of hybrids. The mean pollen viability of hybrids between B. napus and tetraploid B. rapa (80.6%) was high in comparison with mean pollen viability of triploid hybrids between B. napus and diploid B. rapa. Therefore, the occurrence of tetraploid B. rapa should be taken into consideration when estimating the likelihood of gene flow from oilseed rape to close relatives at the landscape level. Tetraploid B. rapa is a common component of several seed mixtures and establishes feral populations in northwest Germany. Assuming a similar abundance of diploid and tetraploid B. rapa, gene flow from B. napus to tetraploid may be more likely than gene flow to diploid B. rapa.     

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.