An expression cDNA library for suppression cloning in yeast mutants, complementation of a yeast his4 mutant, and EST analysis from the symbiotic basidiomycete Hebeloma cylindrosporum.

An oriented expression library was constructed from the mycelia of the symbiotic model fungus Hebeloma cylindrosporum in the high-level yeast expression vector pDR196. DNA sequencing of approximately 500 expressed sequence tags (ESTs) showed that 15% correspond to known genes, two thirds contain sequences with unknown function, andthe remaining 20% showed no significant similarity to any known genes. The ESTs had a GC content between 44 and 56%, with most of them having a GC content of 52-54%, which could be correlated with GC contents of fungal genes. The library was successfully used to identify the Hebeloma HIS4 gene by functional complementation of a yeast his4 mutant. Thus, the library may serve as a powerful tool for identification and characterization of mycorrhizal genes by EST analysis and for the identification of ectomycorrhizal genes by means of suppression cloning.     

Investigations on heat resistance of spinach leaves

Exposure of spinach plants to high temperature (35° C) increased the heat resistance of the leaves by about 3° C. This hardening process occurred within 4 to 6 h, whereas dehardening at 20°/15° C required 1 to 2 days. At 5° C dehardening did not take place. Hardening and dehardening occurred in both the dark and the light. The hardiness was tested by exposure of the leaves to heat stress and subsequent measurements of chlorophyll fluorescence induction and light-induced absorbance changes at 535 nm on the leaves and of the photosynthetic electron transport in thylakoids isolated after heat treatment. Heat-induced damage to both heat-hardened and non-hardened leaves seemed to consist primarily in a breakdown of the membrane potential of the thylakoids accompanied by partial inactivation of electron transport through photosystem II. The increase in heat resistance was not due to temperature-induced changes in lipid content and fatty acid composition of the thylakoids, and no conspicuous changes in the polypeptide composition of the membranes were observed. Prolonged heat treatment at 35° C up to 3 days significantly decreased the total lipid content and the degree of unsaturation of the fatty acids of membrane lipids without further increase in the thermostability of the leaves. Intact chloroplasts isolated from heat-hardened leaves retained increased heat resistance. When the stroma of the chloroplasts was removed, the thermostability of the thylakoids was decreased and was comparable to the heat resistance of chloroplast membranes obtained from non-hardened control plants. Compartmentation studies demonstrated that the content of soluble sugars within the chloroplasts and the whole leaf tissue decreased as heat hardiness increased. This indicated that in spinach leaves, sugars play no protective role in heat hardiness. The results suggest that changes in the ultrastructure of thylakoids in connection with a stabilizing effect of soluble non-sugar stroma compounds are responsible for acclimatization of the photosynthetic apparatus to high temperature conditions. Changes in the chemical composition of the chloroplast membranes did not appear to play a role in the acclimatization.Abbreviations DGDG digalactosyl diglyceride - MGDG monogalactosyl diglyceride - PG phosphatidyl glycerol - PGA 3-phosphoglyceric acid Dedicated to Professor Wilhelm Simonis, Würzburg, on the occasion of his 70th birthday     

Role of source-to-sink transport of methionine in establishing seed protein quantity and quality in legumes

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Methionine transporter function in pea phloem and embryo affects sulfur, nitrogen, and carbon acquisition, metabolism, and partitioning, resulting in increased seed yield, protein levels, and quality.     

Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3

Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.     

Impaired synapse function during postnatal development in the absence of CALEB, an EGF-like protein processed by neuronal activity

In an attempt to characterize the molecular components by which electric activity influences the development of synapses, we searched for cell surface proteins modulated by calcium influx and glutamate receptor activity. Here, we report that neuronal depolarization facilitates the conversion of CALEB, which results in a truncated transmembrane form with an exposed EGF domain. To characterize the role of CALEB in synapse development, synaptic features were investigated in slices of the colliculus superior from CALEB-deficient mice. In the absence of CALEB, the number of synapses and their morphological characteristics remained unchanged. However, in CALEB-deficient mice, synapses displayed higher paired-pulse ratios, less depression during prolonged repetitive activation, a lower rate of spontaneous postsynaptic currents, and a lower release probability at early but not mature postnatal stages. Our findings indicate that CALEB provides a molecular basis for maintaining normal release probability at early developmental stages.     

The Haloferax volcanii FtsY homolog is critical for haloarchaeal growth but does not require the A domain

The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In Eukarya and Bacteria it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Sralpha/beta and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Sralpha/FtsY homologs. In the present study the FtsY homolog of Haloferax volcanii was characterized in its native host. Disruption of the sole chromosomal copy of ftsY in H. volcanii was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in trans. Subcellular fractionation analysis of H. volcanii ftsY deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both H. volcanii strains. These analyses suggest that the H. volcanii FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.     

Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea

The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.     

Glycosylation of Eag1 (Kv10.1) potassium channels: intracellular trafficking and functional consequences

N-Linked glycosylation is a common post-translational modification of membrane proteins. Here we report that mature Eag1 potassium channels carry sugar moieties linked to asparagines at positions 388 and 406. Asn-388 seems to undergo only core glycosylation, but complex sugars are bound to Asn-406. Correct complex glycosylation is required for proper trafficking of Eag1 to the plasma membrane but is also crucial for the correct function of channels already inserted in the membrane.     

Got Milk? Breastfeeding and Milk Analysis of a Mother on Chronic Hemodialysis

Purpose

Women on dialysis rarely become pregnant. However, the overall rate of successful pregnancies is increasing in this patient population and breastfeeding becomes an option for mothers on dialysis. In this study we performed a systematic breast milk composition analysis of a mother on chronic hemodialysis (HD).

Methods

Specimens of breast milk and blood were collected in regular intervals before and after HD from a 39-year old woman starting on day 10 postpartum. Samples were analyzed for electrolytes, retention solutes, nutrients and other laboratory measurements. Breast milk samples from low-risk mothers matched for postpartum age were used as controls.

Results

Significantly higher levels of creatinine and urea were found in pre-HD breast milk when compared to post-HD. A similar post-dialytic decrease was only found for uric acid but not for any other investigated parameter. Conversely, sodium and chloride were significantly increased in post-HD samples. Compared to controls creatinine and urea were significantly higher in pre-HD samples while the difference remained only significant for post-HD creatinine. Phosphate was significantly lower in pre- and post-HD breast milk when compared to controls, whereas calcium showed no significant differences. In terms of nutrient components glucose levels showed a strong trend for a decrease, whereas protein, triglycerides and cholesterol did not differ. Similarly, no significant differences were found in iron, potassium and magnesium content.

Conclusion

To the best of our knowledge this is the first report on a breastfeeding mother on chronic dialysis. Although we found differences in creatinine, urea, sodium, chloride and phosphate, our general analysis showed high similarity of our patient’s breast milk to samples from low-risk control mothers. Significant variations in breast milk composition between pre- and post-HD samples suggest that breastfeeding might be preferably performed after dialysis treatment. In summary, our findings indicate that breastfeeding can be considered a viable option for newborns of mothers on dialysis.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.