Identification and characterization of the mouse nuclear export factor (Nxf) family members

TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm.     

The 29.9 kDa subunit of mitochondrial complex I is involved in the enzyme active/de-active transitions

Mitochondrial respiratory chain complex I undergoes transitions from active to de-activated forms. We have investigated the phenomenon in sub-mitochondrial particles from Neurospora crassa wild-type and a null-mutant lacking the 29.9 kDa nuclear-coded subunit of complex I. Based on enzymatic activities, genetic crosses and analysis of mitochondrial proteins in sucrose gradients, we found that about one-fifth of complex I with catalytic properties similar to the wild-type enzyme is assembled in the mutant. Mutant complex I still displays active/de-active transitions, indicating that other proteins are involved in the phenomenon. However, the kinetic characteristics of complex I active/de-active transitions in nuo29.9 differ from wild-type. The spontaneous de-activation of the mutant enzyme is much slower, implicating the 29.9 kDa polypeptide in this event. We suggest that the fungal 29.9 kDa protein and its homologues in other organisms may modulate the active/de-active transitions of complex I.     

A three-dimensional cryo-electron microscopy structure of the bacteriophage phiKZ head

The three-dimensional structure of the Pseudomonas aeruginosa bacteriophage phiKZ head has been determined by cryo-electron microscopy and image reconstruction to 18A resolution. The head has icosahedral symmetry measuring 1455 A in diameter along 5-fold axes and a unique portal vertex to which is attached an approximately 1800 A-long contractile tail. The 65 kDa major capsid protein, gp120, is organized into a surface lattice of hexamers, with T = 27 triangulation. The shape and size of the hexamers is similar to the hexameric building blocks of the bacteriophages T4, phi29, P22, and HK97. Pentameric vertices of the capsid are occupied by complexes composed of several special vertex proteins. The double-stranded genomic DNA is packaged into a highly condensed series of layers, separated by 24 A, that follow the contour of the inner wall of the capsid.     

Comparison of the Munich Chronotype Questionnaire with the Horne-Ostberg's Morningness-Eveningness Score

We report on results from an Internet survey of sleeping habits in a Dutch population using the Munich Chronotype Questionnaire (MCTQ), supplemented with the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ). The MCTQ was completed by 5,055 responders, of which 2,481 also completed the MEQ. MEQ score correlated well with the MCTQ assessment of time of mid-sleep on free days (MSF; r = - 0.73) and on workdays (MSW; r = - 0.61). MEQ was more strongly correlated with MSF (50% of sleep time) than with sleep onset (0%), rise time (100%), or with any other percentile (10 to 40, 60% to 90%) of sleep on free days. The study shows that chronotype (based on MSF as measured by the MCTQ) strongly correlates with morningness-eveningness (as measured by the MEQ). However, the MCTQ collects additional detailed information on sleep-wake behavior under natural conditions.     

Estimating the life-span of oligodendrocytes from clonal data on their development in cell culture

This paper presents a new method to analyze clonal data on oligodendrocyte development in cell culture. The process of oligodendrocyte generation from precursor cells is modelled as a multi-type Bellman-Harris branching process as suggested in an earlier paper [K. Boucher, A. Zorin, A.Y. Yakovlev, M. Mayer-Proschel, M. Noble, An alternative stochastic model of generation of oligodendrocytes in cell culture, J. Math. Biol. 43 (2001) 22]. This model has been extended to allow for death of oligodendrocytes as well as a dissimilar distribution of the first mitotic cycle duration as compared to the subsequent cycles of precursor cells, which lengths are assumed to be independent and identically distributed random variables. Since the time-span of oligodendrocytes is not directly observable in clonal data, plausible parametric assumptions are invoked to make estimation problems tractable. In particular, the time to cell death follows a two-parameter gamma distribution, while the lapse of time between the event of cell death and the event of cell disintegration is assumed to be exponentially distributed. A simulated pseudo maximum likelihood method for estimation of model parameters has been developed using simulation-based approximations of the expected numbers and variance-covariance matrices for different types of cells. Finite sample properties of the estimation procedure are studied by computer simulations. The proposed method is illustrated with an analysis of the clonal development of O-2A progenitor cells isolated from the rat optic nerve and the corpus callosum.     

AbrB-like transcription factors assume a swapped hairpin fold that is evolutionarily related to double-psi beta barrels

AbrB is a key transition-state regulator of Bacillus subtilis. Based on the conservation of a betaalphabeta structural unit, we proposed a beta barrel fold for its DNA binding domain, similar to, but topologically distinct from, double-psi beta barrels. However, the NMR structure revealed a novel fold, the "looped-hinge helix." To understand this discrepancy, we undertook a bioinformatics study of AbrB and its homologs; these form a large superfamily, which includes SpoVT, PrlF, MraZ, addiction module antidotes (PemI, MazE), plasmid maintenance proteins (VagC, VapB), and archaeal PhoU homologs. MazE and MraZ form swapped-hairpin beta barrels. We therefore reexamined the fold of AbrB by NMR spectroscopy and found that it also forms a swapped-hairpin barrel. The conservation of the core betaalphabeta element supports a common evolutionary origin for swapped-hairpin and double-psi barrels, which we group into a higher-order class, the cradle-loop barrels, based on the peculiar shape of their ligand binding site.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

The antioxidant function of the p53 tumor suppressor

It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest or by triggering apoptosis. Here we show that, in addition, p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage and genetic instability. In the absence of severe stresses, relatively low levels of p53 are sufficient for upregulation of several genes with antioxidant products, which is associated with a decrease in intracellular ROS. Downregulation of p53 results in excessive oxidation of DNA, increased mutation rate and karyotype instability, which are prevented by incubation with the antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevented frequent lymphomas characteristic of Trp53-knockout mice, and slowed the growth of lung cancer xenografts deficient in p53. Our results provide a new paradigm for a nonrestrictive tumor suppressor function of p53 and highlight the potential importance of antioxidants in the prophylaxis and treatment of cancer.     

tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p

We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m7G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-Delta, trm82-Delta, and trm8-Delta trm82-Delta double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m7G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m7G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m7G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre-tRNAPhe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m7G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82-Delta strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation.