Accumulation of plant small heat-stress proteins in storage organs

Plant small heat-stress proteins (sHSPs) have been shown to be expressed not only after exposure to elevated temperatures, but also at particular developmental stages such as embryogenesis, microsporogenesis, and fruit maturation. This paper presents new data on the occurrence of sHSPs in vegetative tissues, their tissue-specific distribution, and cellular localization. We have found sHSPs in 1-year-old twigs of Acer platanoides L. and Sambucus nigra L. and in the liana Aristolochia macrophylla Lamk. exclusively in the winter months. In tendrils of Aristolochia, sHSPs were localized in vascular cambium cells. After budding, in spring, these proteins were no longer present. Furthermore, accumulation of sHSPs was demonstrated in tubers and bulbs of Allium cepa L., Amaryllis ( Hippeastrum hybridum hort.), Crocus albiflorus L., Hyacinthus orientalis L., Narcissus pseudonarcissus L., Tulipa gesneriana L., and Solanum tuberosum L. (potato). In potato tubers and bulb scales of Narcissus the stress proteins were localized in the central vacuoles of storage parenchyma cells. In order to obtain more information on a possible functional correlation between storage proteins and sHSPs, the accumulation of both types of protein in tobacco seeds during seed ripening and germination was monitored. The expression of sHSPs and globulins started simultaneously at about the 17th day after anthesis. During seed germination the sHSPs disappeared in parallel with the storage proteins. Furthermore, in embryos of transgenic tobacco plants, which do not contain any protein bodies or storage proteins, no sHSPs were found. Thus, the occurrence of sHSPs in perennial plant storage organs seems to be associated with the presence of storage proteins.     

Infection of cells with replication deficient adenovirus induces cell cycle alterations and leads to downregulation of E2F-1

Gene products of recombinant replication-deficient adenovirus vectors of the first generation (Ad vector) can induce cell cycle dysregulation and apoptosis after infection in eukaryotic cells. The mechanisms underlying this complex process are largely unknown. Therefore, we investigated the regulation of the pRb/E2F-1 complex, which controls transition from G(0)/G(1) to S phase of the cell cycle. As Ad vector infection results in a decrease in the number of cells in G(0)/G(1) phase of the cell cycle, we observed a decline of the pRb protein level and, surprisingly, also a decrease of the E2F-1 protein and mRNA level in infected cell lines. Furthermore, in contrast to the reduction of cells in the G(0)/G(1) phase we observed increased protein levels of p53 and p21 proteins. However, as experiments in p53 deficient cell lines indicated, the decrease of pRb and E2F-1 is independent of p53 and p21 expression. Moreover, results obtained with Rb deficient cell lines indicated that the reduced E2F-1 expression is independent of pRb. These results suggest that Ad vector-induced cell cycle dysregulation is associated with a specific downregulation of E2F-1 independent of Rb and p53 genomic status of cells.     

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4°C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2′-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2′-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.     

Fish Communities in Central Amazonian White- and Blackwater Floodplains

In Amazonian floodplains, the flood cycle of the river is becoming the dominant seasonal factor, and fish communities are found to fluctuate greatly over the year. During inundation, fish migrate into floodplain forests to feed on fruits and seeds, in an area more than 300000km² in size. To document patterns of species diversity, distribution, abundance and temporal dynamics and in order to describe the ecological importance of the inundated forest, floodplain fish were captured using variously sized gill nets in white and black water areas inside and outside the floodplain forests during low, rising, high and falling water level in 1990 and 1991. Dominance varies to some extent in white water between floodplain forest (0.06) and open water (0.11) while it is unchanged in black water (0.04). Black water fish communities were more diverse. Most abundant among white water fish were Liposarcus pardalis, Pygocentrus nattereri, and Pellona flavipinnis, for example, or Plagioscion squamsissimus, Serrasalmus rhombeus, and Serrasalmus manueli in black water. Among the most abundant white water fish, Colossoma macropomum, Mylossoma duriventre and Osteoglossum bicirrhosum occurred almost exclusively in inundated forests. Of the black water species there were a large number of species which were captured only in inundated forest, such as Geophagus cf. altifrons, Hoplias malabaricus, Osteoglossum bicirrhosum and Uaru amphiacanthoides. Catches varied with sample site, water level and direction of water level change. The average CPUE in white and black water was 190 and 41g fishm^–2 and day, respectively, with maximum yields at low water and minimum yields at high water. Comparing rising and falling water levels, a significantly higher quantity of fishes was captured at falling water level. In black water, fish catches from the floodplain forest exceeded the open water catch by 183 to 550%, depending on season. Differences in respect of white water are smaller (106–281%). Fish communities in the area under investigation seem to be stochastically assembled, with significant differences between white and black water only. Many fishes move into the floodplain forest not only to feed but probably also for other reasons – to seek shelter, for example.     

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α₁subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ?-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ?-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.     

The characteristics and transparent exopolymer particle (TEP) content of marine snow formed from thecate dinoflagellates

Abundant marine snow containing diatoms and detritus, but dominatedby large, bioluminescent thecate dinoflagellates and their temporaryvegetative cysts, especially several species of the genus Gonyoulax,was observed at six stations in the Santa Barbara Channel, California,in 1989 and 1994. These aggregates were unusually cohesive andmucus rich, and contained 2–4 times more mass, particulateorganic carbon (POC), particulate organic nitrogen (PON) andchlorophyll a per unit aggregate volume than more common typesof marine snow formed from diatoms, fecal matter, larvaceanhouses or miscellaneous detritus. However, the relationshipbetween aggregate size and the concentration of TEP (transparentexopolymer particles which form the mucus matrix of most marinesnow) was similar to that of other types of aggregates, suggestingthat much of the copious gel-like material within dinoflagellateaggregates was not TEP. While this is the first report of abundantthecate dinoflagellates occurring within large, rapidly sinkingmarine aggregates, the data do not support the conclusion thatmass aggregation and subsequent sedimentation of blooms is partof the life history adaptations of thecate dinoflagellates,as it is for some diatoms. The high abundance of free-livingdinoflagellate cells and temporary cysts, and the similar proportionof dinoflagellates relative to other algal and chemical componentsin both aggregates and the surrounding seawater, indicate thatthe dinoflagellates were not differentially aggregating. Evenso, passive accumulation of dinoflagellates in marine snow throughaggregation processes may result in more rapid transport ofdinoflagellate-generated material to the deep ocean, alter thenature of sinking particulate matter following dinoflagellateblooms, and increase the nutritional value of marine snow asa food source for zooplankton and fish.     

Differences in the Serum Nonesterified Fatty Acid Profile of Young Women Associated with a Recent History of Gestational Diabetes and Overweight/Obesity

BackgroundNonesterified fatty acids (NEFA) play pathophysiological roles in metabolic syndrome and type 2 diabetes (T2D). In this study, we analyzed the fasting NEFA profiles of normoglycemic individuals at risk for T2D (women with a recent history of gestational diabetes (GDM)) in comparison to controls (women after a normoglycemic pregnancy). We also examined the associations of NEFA species with overweight/obesity, body fat distribution and insulin sensitivity.ResultsWomen after GDM had a lower molar percentage of total saturated fatty acids (SFA; 38.55% vs. 40.32%, p = 0.0002) than controls. At an explorative level of significance several NEFA species were associated with post-GDM status (with and without adjustment for body mass index (BMI) and HbA1c): The molar percentages of 14:0, 16:0, 18:0 and 18:4 were reduced, whereas those of 18:1, 18:2, 20:2, 24:4, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and total n-6 NEFA were increased. BMI and the amount of body fat correlated inversely with several SFA and MUFA and positively with various PUFA species over the whole study cohort (abs(ρ)≥0.3 for all). 14:0 was inversely and BMI-independently associated with abdominal visceral adiposity. We saw no correlations of NEFA species with insulin sensitivity and the total NEFA concentration was similar in the post-GDM and the control group.ConclusionIn conclusion, we found alterations in the fasting NEFA profile associated with a recent history of gestational diabetes, a risk marker for T2D. NEFA composition also varied with overweight/obesity and with body fat distribution, but not with insulin sensitivity.     

Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.     

Development of a Novel Strategy to Isolate Lipophilic Allergens (Oleosins) from Peanuts

Background

Peanut allergy is one of the most severe class I food allergies with increasing prevalence. Especially lipophilic allergens, such as oleosins, were found to be associated with severe symptoms, but are usually underrepresented in diagnostic extracts. Therefore, this study focused on isolation, molecular characterization and assessment of the allergenicity of peanut oleosins.

Methods and Results

A comprehensive method adapted for the isolation of peanut oil bodies of high purity was developed comprising a stepwise removal of seed storage proteins from oil bodies. Further separation of the oil body constituents, including the allergens Ara h 10, Ara h 11, the presumed allergen oleosin 3 and additional oleosin variants was achieved by a single run on a preparative electrophoresis cell. Protein identification realized by N-terminal sequencing, peptide mass fingerprinting and homology search revealed the presence of oleosins, steroleosins and a caleosin. Immunoblot analysis with sera of peanut-allergic individuals illustrated the IgE-binding capacity of peanut-derived oleosins.

Conclusion

Our method is a novel way to isolate all known immunologically distinct peanut oleosins simultaneously. Moreover, we were able to provide evidence for the allergenicity of oleosins and thus identified peanut oleosins as probable candidates for component-resolved allergy diagnosis.