Membranfluß und Nucleosiddiphosphatase-Reaktion in Wurzelhaaren von Lepidium sativum

Summary Root hairs of Lepidium sativum were incubated with a Wachstein-Meisel medium in experiments designed to localize the activity of nucleoside diphosphatase(s). Electron dense precipitates were found in the ER and in Golgi cisternae of the secretory face of the dictyosomes and their adjacent Golgi vesicles. Such precipitates were absent in the Golgi cisternae of the regeneration face of the dictyosomes and in the detached Golgi vesicles which extrude pectic cell wall substances. These results may be the consequence of the normal cycle of membrane compounds associated with the secretion in which the nucleoside diphosphatase(s) participate (by activation and inactivation) as one of the cycling components. Alternatively the nucleoside diphosphatase(s) may undergo a special cycle in which they are transferred from one cisterna or its vesicles to the next as part of the process of cisternal maturation.     

Dorsiventralität der Statocyten in plagiogeotropen Seitenwurzeln von Lepidium sativum L.

Summary The calyptra of plagiotropic lateral roots of Lepidium sativum L. is composed of three rows of cells. Movable amyloplates, possibly functioning as statoliths, are located only a few central cells of the ontogenetic youngest cell row. Beside the lateral root axis the two innermost statocytes contain a stable complex of rough endoplasmic reticulum, which is preferentially located in the central distal cell corner. In the statocytes lying above the lateral root axis the amyloplasts are sedimented on the ER-complex during growth in direction of the geotropic liminal angle. In the statocyte below the axis the ER-complex is free of amyloplasts. Thus a dorsiventrality exists in the statocytes located above and below the root axis in regard to the arrangement of their organelles.

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Herrn Professor Dr. Maximilian Steiner zum 70. Geburtstag.     

Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate–protein ligase A

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate–protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.     

Features of protein−protein interactions in the cyanobacterial photoprotection mechanism

Photoprotective mechanisms of cyanobacteria are characterized by several features associated with the structure of their water-soluble antenna complexes–the phycobilisomes (PBs). During energy transfer from PBs to chlorophyll of photosystem reaction centers, the “energy funnel” principle is realized, which regulates energy flux due to the specialized interaction of the PBs core with a quenching molecule capable of effectively dissipating electron excitation energy into heat. The role of the quencher is performed by ketocarotenoid within the photoactive orange carotenoid protein (OCP), which is also a sensor for light flux. At a high level of insolation, OCP is reversibly photoactivated, and this is accompanied by a sig- nificant change in its structure and spectral characteristics. Such conformational changes open the possibility for pro- tein–protein interactions between OCP and the PBs core (i.e., activation of photoprotection mechanisms) or the fluores- cence recovery protein. Even though OCP was discovered in 1981, little was known about the conformation of its active form until recently, as well as about the properties of homologs of its N and C domains. Studies carried out during recent years have made a breakthrough in understanding of the structural-functional organization of OCP and have enabled discovery of new aspects of the regulation of photoprotection processes in cyanobacteria. This review focuses on aspects of protein–pro- tein interactions between the main participants of photoprotection reactions and on certain properties of representatives of newly discovered families of OCP homologs.     

SHARP: genome-scale identification of gene–protein–reaction associations in cyanobacteria

Genome scale metabolic model provides an overview of an organism’s metabolic capability. These genome-specific metabolic reconstructions are based on identification of gene to protein to reaction (GPR) associations and, in turn, on homology with annotated genes from other organisms. Cyanobacteria are photosynthetic prokaryotes which have diverged appreciably from their nonphotosynthetic counterparts. They also show significant evolutionary divergence from plants, which are well studied for their photosynthetic apparatus. We argue that context-specific sequence and domain similarity can add to the repertoire of the GPR associations and significantly expand our view of the metabolic capability of cyanobacteria. We took an approach that combines the results of context-specific sequence-to-sequence similarity search with those of sequence-to-profile searches. We employ PSI-BLAST for the former, and CDD, Pfam, and COG for the latter. An optimization algorithm was devised to arrive at a weighting scheme to combine the different evidences with KEGG-annotated GPRs as training data. We present the algorithm in the form of software “Systematic, Homology-based Automated Re-annotation for Prokaryotes (SHARP).” We predicted 3,781 new GPR associations for the 10 prokaryotes considered of which eight are cyanobacteria species. These new GPR associations fall in several metabolic pathways and were used to annotate 7,718 gaps in the metabolic network. These new annotations led to discovery of several pathways that may be active and thereby providing new directions for metabolic engineering of these species for production of useful products. Metabolic model developed on such a reconstructed network is likely to give better phenotypic predictions.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ø29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^?) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.     

Accumulation of lead in root cells of Pisum sativum

The ever-increasing environmental pollution necessitates organisms to develop specific defense systems in order to survive and function effectively. Lead is taken up by plants mainly through roots and over 96% are accumulated there.Pea plants were cultivated hydroponically for 4 days with 0.1, 0.5 and 1 mM Pb(NO₃)₂. Uptake of lead ions from nutrient solution and accumulation in root stems and leaves during 96-h cultivation was estimated. The root tip cells were observed with transmission electron microscope to analyse their ultrastructure and lead localization. Pb was accumulated in the cell wall, cell membrane, vacuoles, mitochondria and peroxisomes. The fractions of mitochondria and peroxisomes were isolated from pea roots purified by means Percoll gradient, and were observed by means of electron microscope with the attachment for X-ray microanalysis. Visible deposits containing Pb were observed in both cell organelles.     

Expression profile of the α subunit of the heterotrimeric G protein in rice

Previous studies on the activity of the rice Gα promoter using a β-Glucuronidase (GUS) reporter construct indicated that Gα expression was highest in developing organs and changed in a developmental stage-dependent manner. In this paper, GUS activity derived from the rice Gα promoter was analyzed in seeds and developing leaves. In seeds, GUS activity was detected in the aleurone layer, embryo, endosperm and scutellar epithelium. In developing leaves, the activity was detected in the mesophyll tissues, phloem and xylem of the leaf sheath and in the mesophyll tissue of the leaf blade. The activity in the aleurone layer and scutellar epithelium suggests that the Gα subunit may be involved in gibberellin signaling. The activity in the mesophyll tissues of the leaf blade suggests that the Gα subunit may be related to the intensity of disease resistance. The pattern of the activity in the developing leaf also indicates that the expression of Gα follows a developmental profile at the tissue level.Key words: expression pattern, Gα subunit, GUS staining pattern, heterotrimeric G protein, riceThe rice mutant d1 is deficient in the heterotrimeric G protein α subunit (Gα). Recently it was found that the dwarfism phenotype of d1 is due to a reduction in cell numbers.¹ This discovery has led to new questions regarding how rice Gα regulates cell number, and which other signaling molecules are involved in this process in various tissues and at different development stages. Studies of d1 suggest that rice Gα participates in both gibberellin signaling^2–4 and brassinosteroid signaling.^5–8 Promoter studies using the β-Glucuronidase (GUS) reporter indicate that Gα expression is highest in developing organs.¹ In this paper, we report on the expression pattern of a Gα promoter::GUS construct in seeds and developing leaves of rice.     

Protein–protein interactions of huntingtin in the hippocampus

Molecular Biology - Huntingtin (HTT) occurs in the neuronal cytoplasm and can interact with structural elements of synapses. Huntington’s disease (HD) results from pathological expansion of a...     

Advances in the study of protein–DNA interaction

Protein-DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein-DNA. Recent developments of these methods and research achievements have been reviewed in this paper.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Transglycosylation of a β-galactosyl radical, in the course of enzymic hydrolysis of lactose, in the presence of selected polyhydroxyalcohols

Sorbitol, xylitol, erythritol and lactitol were used as the acceptors of galactosyl radicals, in the process of transgalactosylation accompanying the hydrolysis of lactose, conducted with -galactosidase (4.0 ml Lactozym 3000 was added to 430 ml 1.45 M lactose with 0.95 M polyhydroxyalcohols). The following concentrations of galactosyl derivatives of polyols were obtained after hydrolysis for 4 h at 40°C: 0.31 M Gal-erythritol, 0.22 M Gal-xylitol, 0.18 M Gal-sorbitol and 0.14 M Gal-lactitol. A quadruple increase of xylitol content in dry matter of a solution (from 11.5% (w/w) to 44.5% (w/w) brought about a 2.3-fold increase of the product content in the solution (15.2% (w/w) of dry matter).     

Nuclear protein in the liver in protein–calorie deficiency

1. The effect of protein–calorie deficiency on nuclear proteins was studied in growing and adult rats by using chemical and electrophoretic methods of fractionation. 2. After 7 days on protein-deficient diets, the amount of neutral-soluble proteins increased, and their electrophoretic pattern changed, with the appearance of a new component. 3. The histone fraction of the liver nuclei in rapidly growing rats was lower than that in either deficient or adult animals. 4. The possible role of nuclear proteins in relation to synthesis of the proteins and nucleic acids of the other parts of the cells is discussed.     

Evaluation of the Vitek-MS™ system in the identification of Candida isolates from bloodstream infections

Background

Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) represents a revolution in the identification of microorganisms of clinical interest. Many studies have confirmed the accuracy and fastness of this tool with routine strains.

High-throughput docking for the identification of new influenza A virus polymerase inhibitors targeting the PA–PB1 protein–protein interaction

A high-throughput molecular docking approach was successfully applied for the selection of potential inhibitors of the Influenza RNA-polymerase which act by targeting the PA–PB1 protein–protein interaction. Commercially available compounds were purchased and biologically evaluated in vitro using an ELISA-based assay. As a result, some compounds possessing a 3-cyano-4,6-diphenyl-pyridine nucleus emerged as effective inhibitors with the best ones showing IC₅₀ values in the micromolar range.     

Increased yield of β-glucosidase-catalyzed hydrolysis reactions in the presence of betaine-type metabolite analog

β-Glucosidases (EC 3.2.1.21), abundant enzymes distributed in animals, plants and microorganism, has been generating lots of attentions for bioethanol production from cellulosic biomass. In this study, using three different origins of β-glucosidases, glucose productivity of β-glucosidase-catalyzed hydrolysis reactions in the presence of synthetic betaine-type metabolite analog (2-N,N,N-tri-n-butylammonium) acetate, was investigated. By the addition of the analog, the hydrolysis yields for all β-glucosidases was highly improved from 4–13 to 64–100 %. To understand the factors affecting on the yield enhancements, the kinetic parameters, inhibition constants of end-product and temporal stability of β-glucosidases were compared. As a result, enhancement of the yields is mainly related to the increase in the temporal stability of β-glucosidases in the presence of the analog. The present findings lead to not only improve the glucose productivity of β-glucosidase-catalyzed hydrolysis reaction toward bioethanol production but also apply to a new stabilization method for various unstable enzymes.     

The ∈ subunit of the chloroplast ATP synthase of Pisum sativum

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea or subunit or both. The pea subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in (CF1-). Spinach inhibited the ATPase activity of pea CF1- at lower concentrations than pea . The gene encoding the pea subunit was cloned and over-expressed. Recombinant pea did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-, although pea was effective when tested with pea thylakoids reconstitituted with pea CF1-. These results confirm earlier suggestions that the C-terminal region of is important in -CF1 and -CFo interactions.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ?29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^–) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.