Vergleichende Permeabilitätsstudien an Pflanzenzellen

Ohne ZusammenfassungHerrn Privatdozent Dr. Czaja, auf dessen Veranlassung die Arbeit im Pflanzenphysiologischen Institut der Universität Berlin in Berlin-Dahlem unternommen wurde, bin ich zu herzlichem Dank für seine fördernden Ratschläge verpflichtet.Ebenso danke ich Herrn Direktor Professor Dr. Noack verbindlich für sein Interesse an der Arbeit und GewÄhrung vieler Hilfsmittel.     

Swelling activation of transport pathways in erythrocytes: effects of Cl-, ionic strength, and volume changes

If swelling of acell is induced by a decrease in external medium tonicity, theregulatory response is more complex than if swelling of similarmagnitude is due to salt uptake. The present results provide anexplanation. In fish erythrocytes, two distinct transport pathways wereswelling activated: a channel of broad specificity and aK⁺-Clcotransporter. Each was activated by a specific signal: the channel bya decrease in intracellular ionic strength and theK⁺-Clcotransporter by cell enlargement. A decrease in ionic strength alsoaffectedK⁺-Clcotransport activity, but by acting as a negative modulator of thecotransport. Thus cells swollen by salt accumulation respond byactivating exclusively theK⁺-Clcotransport, leading to aCl-dependentK⁺ loss. By contrast, cellsswollen by electrolyte dilution respond by activating both pathways,leading to a reduced loss of electrolytes and a large loss of taurine.Thus two swelling-sensitive pathways, differently regulated, wouldallow control of the ionic composition of a cell exposed to differentvolume perturbations.

     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

GenoFrag: software to design primers optimized for whole genome scanning by long-range PCR amplification

Genome sequence data can be used to analyze genome plasticity by whole genome PCR scanning. Small sized chromosomes can indeed be fully amplified by long-range PCR with a set of primers designed using a reference strain and applied to several other strains. Analysis of the resulting patterns can reveal the genome plasticity. To facilitate such analysis, we have developed GenoFrag, a software package for the design of primers optimized for whole genome scanning by long-range PCR. GenoFrag was developed for the analysis of Staphylococcus aureus genome plasticity by whole genome amplification in ~10 kb-long fragments. A set of primers was generated from the genome sequence of S.aureus N315, employed here as a reference strain. Two subsets of primers were successfully used to amplify two portions of the N315 chromosome. This experimental validation demonstrates that GenoFrag is a robust and reliable tool for primer design and that whole genome PCR scanning can be envisaged for the analysis of genome diversity in S.aureus, one of the major public health concerns worldwide.     

Fibroblasts form a body-wide cellular network

Loose connective tissue forms a network extending throughout the body including subcutaneous and interstitial connective tissues. The existence of a cellular network of fibroblasts within loose connective tissue may have considerable significance as it may support yet unknown body-wide cellular signaling systems. We used a combination of histochemistry, immunohistochemistry, confocal scanning laser microscopy (confocal microscopy), and electron microscopy to investigate the extent and nature of cell-to-cell connections within mouse subcutaneous connective tissue. We found that fibroblasts formed a reticular web throughout the tissue. With confocal microscopy, 30% of fibroblasts processes could be followed continuously from one cell to another. Connexin 43 immunoreactivity was present at apparent points of cell-to-cell contact. Electron microscopy revealed that processes from adjacent cells were in close apposition to one another, but gap junctions were not observed. Our findings indicate that soft tissue fibroblasts form an extensively interconnected cellular network, suggesting they may have important and so far unsuspected integrative functions at the level of the whole body.     

Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds

We studied the influence of the internal oxygen concentration in seeds of wheat (Triticum aestivum) on storage metabolism and its relation to phloem import of nutrients. Wheat seeds that were developing at ambient oxygen (21%) were found to be hypoxic (2.1%). Altering the oxygen supply by decreasing or increasing the external oxygen concentration induced parallel changes in the internal oxygen tension. However, the decrease in internal concentration was proportionally less than the reduction in external oxygen. This indicates that decreasing the oxygen supply induces short-term adaptive responses to reduce oxygen consumption of the seeds. When external oxygen was decreased to 8%, internal oxygen decreased to approximately 0.5% leading to a decrease in energy production via respiration. Conversely, increasing the external oxygen concentration above ambient levels increased the oxygen content as well as the energy status of the seeds, indicating that under normal conditions the oxygen supply is strongly limiting for energy metabolism in developing wheat seeds. The intermediate metabolites of seed storage metabolism were not substantially affected when oxygen was either increased or decreased. However, at subambient external oxygen concentrations (8%) the metabolic flux of carbon into starch and protein, measured by injecting (14)C-Suc into the seeds, was reduced by 17% and 32%, respectively, whereas no significant effect was observed at superambient (40%) oxygen. The observed decrease in biosynthetic fluxes to storage compounds is suggested to be part of an adaptive response to reduce energy consumption preventing excessive oxygen consumption when oxygen supply is limited. Phloem transport toward ears exposed to low (8%) oxygen was significantly reduced within 1 h, whereas exposing ears to elevated oxygen (40%) had no significant effect. This contrasts with the situation where the distribution of assimilates has been modified by removing the lower source leaves from the plant, resulting in less assimilates transported to the ear in favor of transport to the lower parts of the plant. Under these conditions, with two strongly competing sinks, elevated oxygen (40%) did lead to a strong increase in phloem transport to the ear. The results show that sink metabolism is affected by the prevailing low oxygen concentrations in developing wheat seeds, determining the import rate of assimilates via the phloem.     

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.     

Microscopy images as interactive tools in cell modeling and cell biology education

The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of structures and functions of living cells, to introduce people to the scientific method, to stimulate inquiry, and to analyze and synthesize concepts and paradigms. In this essay we present our experience in mixing science and education in Brazil. For two decades we have developed activities for the science education of teachers and undergraduate students, using microscopy images generated by our work as cell biologists. We describe open-air outreach education activities, games, cell modeling, and other practical and innovative activities presented in public squares and favelas. Especially in developing countries, science education is important, since it may lead to an improvement in quality of life while advancing understanding of traditional scientific ideas. We show that teaching and research can be mutually beneficial rather than competing pursuits in advancing these goals.     

Regulation of DNA replication by ATR: signaling in response to DNA intermediates

The nuclear protein kinase ATR controls S-phase progression in response to DNA damage and replication fork stalling, including damage caused by ultraviolet irradiation, hyperoxia, and replication inhibitors like aphidicolin and hydroxyurea. ATR activation and substrate specificity require the presence of adapter and mediator molecules, ultimately resulting in the downstream inhibition of the S-phase kinases that function to initiate DNA replication at origins of replication. The data reviewed strongly support the hypothesis that ATR is activated in response to persistent RPA-bound single-stranded DNA, a common intermediate of unstressed and damaged DNA replication and metabolism.