CugP Is a Novel Ubiquitous Non-GalU-Type Bacterial UDP-Glucose Pyrophosphorylase Found in Cyanobacteria

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate and exists in almost all species. Most bacteria possess a GalU-type UDP-glucose pyrophosphorylase, whereas many cyanobacteria species do not. In certain cyanobacteria, UDP-glucose is used as a substrate for synthesis of exopolysaccharide cellulose in spite of the absence of GalU-type UDP-glucose pyrophosphorylase. Therefore, there should be an uncharacterized UDP-glucose pyrophosphorylase in cyanobacteria. Here, we show that all cyanobacteria possess a non-GalU-type bacterial UDP-glucose pyrophosphorylase, i.e., CugP, a novel family in the nucleotide triphosphate transferase superfamily. The expressed recombinant Synechocystis sp. strain PCC 6803 CugP had pyrophosphorylase activity that was highly specific for UTP and glucose 1-phosphate. The fact that the CugP gene cannot be deleted completely in Synechocystis sp. PCC 6803 suggests its central role as the substrate supplier for galactolipid synthesis. Galactolipids are major constituents of the photosynthetic thylakoid membrane and important for photosynthetic activity. Based on phylogenetic analysis, this CugP-type UDP-glucose pyrophosphorylase may have recently been horizontally transferred to certain noncyanobacteria.     

Developmental Expression of the Outer Hair Cell Motor Prestin in the Mouse

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C _v), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C _v was about 70% of the mature level. C _v saturated at P18 when hearing shows full maturation. On the other hand, C _lin, which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor’s operating voltage midpoint, V _pkcm, tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V _pkcm reached a steady-state level near −67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V _pkcm, may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.     

Comparison of rNIS and hNIS as reporter genes for noninvasive imaging of bone mesenchymal stem cells transplanted into infarcted rat myocardium

The purpose of this study was to investigate and compare the feasibility of rat sodium iodide symporter (rNIS) and human sodium iodide symporter (hNIS) as reporter genes for noninvasive monitoring of rat bone marrow mesenchymal stem cells (rBMSCs) transplanted into infarcted rat myocardium. rBMSCs were isolated from rat bone marrow. Adenovirus (Ad) was reconstructed to contain rNIS-enhanced green fluorescent protein (eGFP) or hNIS-eGFP. The transfection efficiency of Ad/eGFP/rNIS and Ad/eGFP/hNIS to rBMSCs was measured by real-time polymerase chain reaction, flow cytometry, Western blot, and immunofluorescence staining. The transfected rBMSCs were transplanted into infarcted rat myocardium followed by a single-photon emission computed tomography (SPECT) study with (99m)Tc-pertechnetate as the radiotracer and by autoradiography. The isolated rBMSCs were CD29, CD44, and CD90 positive and CD34, CD45, and CD11b negative. The expression of rNIS and hNIS in the transfected rBMSCs at both gene and protein levels was obviously higher than that without transfection. The myocardium of rats transplanted with transfected rBMSCs could be visualized by SPECT owing to the accumulation of (99m)Tc-pertechnetate in rBMSCs mediated by exogenous NIS genes. The accumulation of (99m)Tc-pertechnetate in myocardium mediated by rNIS was higher than that by hNIS, which was also confirmed by autoradiography. Both rNIS and hNIS are useful reporter genes to monitor BMSCs transplanted into infarcted myocardium in vivo with rNIS being superior to hNIS as the reporter gene.     

Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.     

LIP19, a basic region leucine zipper protein, is a Fos-like molecular switch in the cold signaling of rice plants

The rice low-temperature-induced lip19 gene encodes a 148-amino-acid basic region/leucine zipper (bZIP) protein, termed LIP19. In this study we characterized LIP19 and showed that it lacks the usual ability of bZIP proteins to homodimerize and to bind DNA, as does the Fos protein in mammals. Using a yeast two-hybrid system, the cDNA clones whose products interact with LIP19 were screened. This search revealed a clone termed OsOBF1 (Oryza sativa OBF1) that encodes a new bZIP protein (OsOBF1). This protein forms a homodimer and binds to the hexamer motif sequence (5'-ACGTCA-3'). The protein-protein interaction in homo- and hetero-combinations between LIP19 and OsOBF1 was confirmed in vitro and in planta. LIP19 and OsOBF1 most likely interact with each other more strongly than OsOBF1 interacts with itself, and the resulting heterodimer binds to the C/G hybrid sequence but not to the hexamer sequence. Whereas the expression patterns of lip19 and OsOBF1 in response to low temperatures were totally opposite, the locations of their expression were almost identical. Based upon the presented data, we propose a model describing the low-temperature signal switching mediated by LIP19 in rice.     

Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24-1

An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.