The dynamical structure of the RNA in alfalfa mosaic virus studied by 31P-nuclear magnetic resonance

The structure of the viral RNA in alfalfa mosaic virus (AlMV) was investigated by means of 31P-nuclear magnetic resonance (NMR). It was found that the 31P-NMR line width of AlMV Top a particles is significantly smaller than that of the larger Bottom particles. At low temperatures, the totational correlation time of the 31P nuclei essentially equals the tumbling rate of the virus particle, indicating that the RNA is contained rigidly inside the virion. At more elevated temperatures, the NMR line width sharpens more than expected on the basis of viscosity changes and the RNA exhibits internal mobility. The occurrence of internal mobility is paralleled by an increased internal mobility of the N-terminal part of the coat protein, as could be observed by 1H-NMR spectroscopy. The influence of EDTA on the 31P-NMR line width appeared to be negligible, which is in agreement with the idea that AlMV does not 'swell' like several other RNA-containing plant viruses.     

Understanding Performance Degradation in Cation‐Disordered Rock‐Salt Oxide Cathodes

The collective redox activities of transition‐metal (TM) cations and oxygen anions have been shown to increase charge storage capacity in both Li‐rich layered and cation‐disordered rock‐salt cathodes. Repeated cycling involving anionic redox is known to trigger TM migration and phase transformation in layered Li‐ and Mn‐rich (LMR) oxides, however, detailed mechanistic understanding on the recently discovered Li‐rich rock‐salt cathodes is largely missing. The present study systematically investigates the effect of oxygen redox on a Li_1.3Nb_0.3Mn_0.4O₂ cathode and demonstrates that performance deterioration is directly correlated to the extent of oxygen redox. It is shown that voltage fade and hysteresis begin only after initiating anionic redox at high voltages, which grows progressively with either deeper oxidation of oxygen at higher potential or extended cycling. In contrast to what is reported on layered LMR oxides, extensive TM reduction is observed but phase transition is not detected in the cycled oxide. A densification/degradation mechanism is proposed accordingly which elucidates how a unique combination of extensive chemical reduction of TM and reduced quality of the Li percolation network in cation‐disordered rock‐salts can lead to performance degradation in these newer cathodes with 3D Li migration pathways. Design strategies to achieve balanced capacity and stability are also discussed.     

Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.     

Two structural genes on different chromosomes are required for encoding the major subunit of human red cell glucose-6-phosphate dehydrogenase

Structural analysis revealed the existence of two types of subunits in human red cell glucose-6-phosphate dehydrogenase. The two subunits have the same COOH region consisting of 479 amino acid residues, but their NH2-terminal regions are different in size and sequence. The minor subunit can be fully encoded by the X-linked G6PD cDNA, but the NH2-terminal region of the major subunit cannot. The cDNA and the gene for the NH2-terminal region of the major subunit were cloned and characterized. Southern blot hybridization indicated that the gene for the NH2-terminal region is on chromosome 6, not on the X chromosome. Northern blot hybridization demonstrated an existence of two separate mRNA components, one for the COOH-terminal region and the other for the NH2-terminal region. Two separate structural genes, the X-linked and chromosome 6-linked genes, must be coresponsible for encoding the single chain subunit. Either cross-translation of two mRNAs, or transpeptidation, or some other mechanism must be involved in the synthesis of human red cell G6PD.     

Primary photosynthetic processes in Heliobacterium chlorum at 15K

Absorbance changes induced by 25-ps laser flashes were measured in membranes of Heliobacterium chlorum at 15 K. Absorbance difference spectra, measured at various times after the flash showed negative bands in the Q_y region at 812, 793 and 665 nm. The first of these bands was attributed to the formation of excited singlet states of a long-wavelength form of antenna bacteriochlorophyll g (BChl g 808). Absorbance changes of shorter wavelength absorbing antenna BChls g were at least an order of magnitude smaller, indicating rapid excitation energy transfer (i.e. within the time resolution of the apparatus) from these BChls to BChl g 808. Excited BChl g 808 showed a bi-exponential decay with time constants of 50 and 200 ps. The bands at 793 and 665 nm may be attributed to the primary charge separation and reflect the photooxidation of the primary electron donor P-798 and photoreduction of a primary electron acceptor absorbing near 670 nm, presumably a BChl c or Chl a-like pigment. The bleaching of this pigment reversed with a time constant of 300 ps at 15 K and of 800 ps at 300 K. This indicates that electron transfer from the primary to the secondary electron acceptor is approximately 2.5 times faster at 15 K than at room temperature.Abbreviations BChl bacteriochlorophyll - FWHM full width at half maximum - P-798 primary electron donor - Tris tris(hydroxymethyl)amino methane     

Structure of tobacco genes encoding thaumatin-like proteins

Two tobacco genes encoding thaumatin-like proteins were cloned and sequenced. Both genes are expressed after infection of tobacco with tobacco mosaic virus (TMV). Comparison of the upstream sequences of these genes with those of other TMV-inducible tobacco genes revealed limited regions of homology.     

Beta-thalassemia mutations in Indonesia and their linkage to beta haplotypes.

A total of 72 chromosomes from 36 Indonesian patients, 23 with beta-thalassemia major and 13 with Hb E-beta-thalassemia, were analyzed by specific oligonucleotide hybridization after DNA amplification. Thirteen had the beta E mutation (codon 26 GAG----AAG). Of the 59-beta-thalassemic chromosomes, 32 were of the variant IVS-1 nt5 (G----C). Seven had the mutation IVS-2 nt654 (C----T), one had the mutation codon 41/42 (deletion CTTT), and one had the mutation codon 17 (AAG----TAG). Another six with the mutation IVS-1 nt1 (G----T), one with the mutation IVS-1 nt1 (G----A), four with the mutation codon 15 (TGG----TAG), one with a mutation codon 30 (AGG----ACG), and one with a mutation codon 35 (deletion C) were first identified by direct sequencing of a patient's genomic DNA followed by further hybridizing other patients' DNA with the appropriate oligonucleotide probes. Five did not carry the common mutations previously described in Asian populations. The four most prevalent mutations encountered made up 83% of the total number of beta-thalassemic chromosomes studied. The most common mutation, IVS-1 nt5 (G----C), was mostly associated with two different haplotypes.     

Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C

Stable expression of high levels of activated forms of Haras (T24) or v-Ki-ras by transfection of Chinese hamster lung fibroblasts (CCL39) yielded cells highly tumorigenic in nude mice. Two classes of transformed cells were distinguished, one with moderate p21 expression (10-fold increased) had retained growth factor dependency, the second with higher level of p21 (greater than 50-fold) appeared autonomous for growth. Neither class of transformants expressing Ki-ras or Ha-ras displayed a significant basal activity of polyphosphoinositide-specific phospholipase C, measured either in serum-starved cells or during exponential growth in the presence of growth factors of the tyrosine kinase family (EGF, FGF, insulin). In the growth-factor-dependent class of T24-Ha-ras-transfected cells (clone 39THaB), phospholipase C could be stimulated normally by serum, thrombin and AlF-4. In the more growth autonomous class (clones 39THaC and 39Ki9), release of inositol phosphates after stimulation with thrombin or serum was drastically reduced. This desensitization, apparently at the receptor level since the response to AlF-4 persisted, is, however, not specific to ras expression. We observed it to the same degree in polyoma virus-transformed CCL39 cells. Finally, expression of mutated forms of p21 ras did not abrogate the sensitivity of phospholipase C activation to pertussis toxin. We conclude that the transforming potential of activated forms of p21ras does not result from persistent activation of phospholipase C and that ras GTP-binding proteins cannot substitute for Gp.