Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

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Highlights? Two structures of inactivated RNRs show two concatenated α₄β₄ rings in the crystal ? Solution scattering shows rings interlock in the presence of crystallization solution ? Electron microscopy shows open and incomplete states of ring formation ? Mechanism of protein ring concatenation by ring opening and closing is proposed     

Arabidopsis ASYMMETRIC LEAVES2 protein required for leaf morphogenesis consistently forms speckles during mitosis of tobacco BY-2 cells via signals in its specific sequence

Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.     

Lack of nocturnal blood pressure fall in elderly bedridden hypertensive patients with cerebrovascular disease

To prevent recurrence of cerebrovascular disease (CVD), adequate control of blood pressure (BP) is extremely important for the treatment of hypertensive CVD patients. As absence of the nocturnal fall of BP by the expected 10-20% from daytime levels is reported to exaggerate target organ injury, 24-h ambulatory blood pressure monitoring (ABPM) was conducted, especially to obtain data during nighttime sleep. Forty-eight elderly bedridden chronic phase CVD hypertensive patients (assessed 1-3 mo after CVD accident) participated. As a group, nocturnal BP was higher than diurnal BP, whereas nocturnal pulse rate was lower than diurnal pulse rate. The nocturnal BP fall was blunted in most (～90%) of the patients. These results suggest that to perform a rational drug treatment, it is essential to do 24-h ABPM before initiation of antihypertensive therapy in elderly bedridden hypertensive CVD patients.     

A novel method for in vitro radiolabeling and testing enveloped viruses by phosphatidylethanolamine N-methyltransferase and host cell-specific binding

The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.     

Studies on the alpha-(1-->4)- and alpha-(1-->8)-fucosylation of sialic acid for the total assembly of the glycan portions of complex HPG-series gangliosides

A synthetic study on alpha-(1-->4) and alpha-(1-->8)-fucosylation of sialic acid is reported, with the ultimate aim being the total assembly of the glycan portion of HPG-series gangliosides. In both types of fucosylations, the combination of a phenylthio fucosyl donor and a 1,5-lactamized acceptor provided high-yielding glycosylations to afford alpha-fucosyl-sialic acid sequences. The obtained alpha-Fucp-(1-->8)-NeupNAc glycan having a 1,5-lactam bridge has been successfully transformed into the corresponding glycosyl donor.     

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.     

Efficient expression of haloarchaeal nucleoside diphosphate kinase via strong porin promoter in moderately halophilic bacteria

Enzymes from extremely halophilic archaea require high concentration of salts for their proper folding and consequently are expressed as an unfolded and inactive form in Escherichia coli. Moderate halophile, which accumulates protein stabilizers, i.e., compatible solutes, is an attractive host cell for the recombinant production of heterologous proteins, since such protein stabilizers may help folding of expressed proteins. Here, we succeeded in efficient expression and purification to homogeneity of recombinant haloarchaeal nucleoside diphosphate kinase (HsNDK) in moderate halophile using newly isolated strong porin promoter.