Changes in distribution of chloroplast adenylate kinase isoforms during floral induction

In the short day plant Chenopodium rubrum and the long day plant Nicotiana tabacum cv. Havana 425, adenylate kinase (EC 2.7.4.3) occurs as a family of isoforms, with at least two members localized in the chloroplast representing the main isoforms. In this work, isoforms were separated by anion exchange chromatography and relative isoform activities were compared between vegetative plants and plants induced to flowering. In both species examined, a light regime leading to floral induction resulted in a significant decrease in the activity of one chloroplast isoform. This decrease modified considerably the relative distribution of isoform activities, especially that between the two chloroplast activities.     

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.     

Stable inheritance of shuttle vectors based on plasmid pIM13 in a mutant strain of Clostridium acetobutylicum.

New shuttle vectors for Clostridium acetobutylicum were constructed, using as replicons the Gram-positive plasmid pIM13, and derivatives of the Gram-negative plasmid pBR322, including pUC19. These vectors transformed C. acetobutylicum at a high frequency (up to 10(6) transformants per microgram DNA) by PEG-mediated protoplast transformation. A mutant host strain, NI-4082, was isolated on the basis of its ability to maintain plasmid pIM13 stably in the absence of selection pressure. The shuttle vectors showed no segregational or structural instability in this mutant strain. Moreover, the results suggested a relationship between segregational instability and the multimerization of pIM13 in C. acetobutylicum. The host/vector system described possessed all the properties required for efficient gene cloning in this species.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectin-like molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.     

A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell

Polarity is often an intrinsic property of the cell, yet little is known about its origin or its maintenance over generations. Here we identify a landmark protein, TipN, which acts as a spatial and temporal cue for setting up the correct polarity in the bacterium Caulobacter crescentus. TipN marks the new pole throughout most of the cell cycle, and its relocation to the nascent poles at the end of division provides a preexisting reference point for orienting the polarity axis in the progeny. Deletion of tipN causes pleiotropic polarity defects, including frequently reversed asymmetry in progeny size and mislocalization of proteins and organelles. Ectopic localization of TipN along the lateral side of the cell creates new axes of polarity leading to cell branching and formation of competent cell poles. Localization defects of the actin-like protein MreB in the DeltatipN mutant suggest that TipN is upstream of MreB in regulating cell polarity.