Requirement for Chloride Channel Function during the Hepatitis C Virus Life Cycle

Hepatocytes express an array of plasma membrane and intracellular ion channels, yet their role during the hepatitis C virus (HCV) life cycle remains largely undefined. Here, we show that HCV increases intracellular hepatic chloride (Cl⁻) influx that can be inhibited by selective Cl⁻ channel blockers. Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl⁻ channel inhibition is detrimental to HCV replication. This represents the first observation of the involvement of Cl⁻ channels during the HCV life cycle.     

Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.     

Rare de novo germline copy-number variation in testicular cancer

Although heritable factors are an important determinant of risk of early-onset cancer, the majority of these malignancies appear to occur sporadically without identifiable risk factors. Germline de novo copy-number variations (CNVs) have been observed in sporadic neurocognitive and cardiovascular disorders. We explored this mechanism in 382 genomes of 116 early-onset cancer case-parent trios and unaffected siblings. Unique de novo germline CNVs were not observed in 107 breast or colon cancer trios or controls but were indeed found in 7% of 43 testicular germ cell tumor trios; this percentage exceeds background CNV rates and suggests a rare de novo genetic paradigm for susceptibility to some human malignancies.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for “respiratory” nitrate reduction ex planta and for nitrogen fixation during symbiosis

We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix^?) and “respiratory” nitrate reduction (Rnr^?). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr^? and Fix^? phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multi-subunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.     

In Vitro Longitudinal Relaxivity Profile of Gd(ABE-DTTA), an Investigational Magnetic Resonance Imaging Contrast Agent

Purpose

MRI contrast agents (CA) whose contrast enhancement remains relatively high even at the higher end of the magnetic field strength range would be desirable. The purpose of this work was to demonstrate such a desired magnetic field dependency of the longitudinal relaxivity for an experimental MRI CA, Gd(ABE-DTTA).

Materials and Methods

The relaxivity of 0.5mM and 1mM Gd(ABE-DTTA) was measured by Nuclear Magnetic Relaxation Dispersion (NMRD) in the range of 0.0002 to 1T. Two MRI and five NMR instruments were used to cover the range between 1.5 to 20T. Parallel measurement of a Gd-DTPA sample was performed throughout as reference. All measurements were carried out at 37°C and pH 7.4.

Results

The relaxivity values of 0.5mM and 1mM Gd(ABE-DTTA) measured at 1.5, 3, and 7T, within the presently clinically relevant magnetic field range, were 15.3, 11.8, 12.4 s^-1mM^-1 and 18.1, 16.7, and 13.5 s^-1mM^-1, respectively. The control 4 mM Gd-DTPA relaxivities at the same magnetic fields were 3.6, 3.3, and 3.0 s^-1mM^-1, respectively.

Conclusions

The longitudinal relaxivity of Gd(ABE-DTTA) measured within the presently clinically relevant field range is three to five times higher than that of most commercially available agents. Thus, Gd(ABE-DTTA) could be a practical choice at any field strength currently used in clinical imaging including those at the higher end.     

Radial glia-like cells at the base of the lateral ventricles in adult mice

During development radial glia (RG) are neurogenic, provide a substrate for migration, and transform into astrocytes. Cells in the RG lineage are functionally and biochemically heterogeneous in subregions of the brain. In the subventricular zone (SVZ) of the adult, astrocyte-like cells exhibit stem cell properties. During examination of the response of SVZ astrocytes to brain injury in adult mice, we serendipitously found a population of cells in the walls of the ventral lateral ventricle (LV) that were morphologically similar to RG. The cells expressed vimentin, glial fibrillary acidic protein (GFAP), intermediate filament proteins expressed by neural progenitor cells, RG and astrocytes. These RG-like cells had long processes extending ventrally into the nucleus accumbens, ventromedial striatum, ventrolateral septum, and the bed nucleus of the stria terminalis. The RG-like cell processes were associated with a high density of doublecortin-positive cells. Lesioning the cerebral cortex did not change the expression of vimentin and GFAP in RG-like cells, nor did it alter their morphology. To study the ontogeny of these cells, we examined the expression of molecules associated with RG during development: vimentin, astrocyte-specific glutamate transporter (GLAST), and brain lipid-binding protein (BLBP). As expected, vimentin was expressed in RG in the ventral LV embryonically (E16, E19) and during the first postnatal week (P0, P7). At P14, P21, P28 as well as in the adult (8–12 weeks), the ventral portion of the LV retained vimentin immunopositive RG-like cells, whereas RG largely disappeared in the dorsal two-thirds of the LV. GLAST and BLBP were expressed in RG of the ventral LV embryonically and through P7. In contrast to vimentin, at later stages BLBP and GLAST were found in RG-like cell somata but not in their processes. Our results show that cells expressing vimentin and GFAP (in the radial glia-astrocyte lineage) are heterogeneous dorsoventrally in the walls of the LV. The results suggest that not all RG in the ventral LV complete the transformation into astrocytes and that the ventral SVZ may be functionally dissimilar from the rest of the SVZ.     

Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor

The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants. In this way, four categories of plants were established. Upon infection with E. coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover. A weak root hair deformation was seen on siratro by inoculation with E. coli harbouring the nodABC genes and was highly increased when hsnD was also introduced. Cowpea and Desmodium did not respond to any of the E. coli strains constructed. Exudates or cytosolicfractions of the respective E. coli derivatives elicited the same root hair deformation as the intact bacteria. These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors. Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover. Coinoculation of alfalfa plants with two strains of E. coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa. These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.     

A subset of clones in the chick telencephalon arranged in rostrocaudal arrays

Background Different areas of the vertebrate central nervous system appear to follow different rules during development for determining the position of sibling cells. For example, in the chick hindbrain, clones are frequently confined to a single functional unit that derives from a single rhombomere. In contrast, clones in the mammalian cerebral cortex often cross functional boundaries because of the extensive migration of sibling cells in orthogonal directions. We have investigated whether the pattern of clonal distribution in the chick telencephalon is similar to that of the hindbrain or to the more functionally analogous mammalian cerebral cortex. Progenitor cells in the chick telencephalon were marked using a retroviral library encoding alkaline phosphatase and over 10⁵ distinct molecular tags. Patterns of dispersion were detected using alkaline phosphatase histochemistry, followed by the recovery and sequencing of the molecular tag. We also analyzed the phenotypes of cells that occurred within the clones.Results A subset of progenitors gave rise to clones that were found in rostrocaudal arrays resembling tubes. Arrays were restricted in the mediolateral and dorsoventral planes but could span up to 4 mm in the rostrocaudal direction. They were found throughout the telencephalon and a single clone often spanned more than one telencephalic nucleus. Rostrocaudal clones comprised 60% of clones containing five or more cells and contained many different types of neurons, astrocytes, oligodendrocytes, or various combinations of these cell types.Conclusions Telencephalic progenitors are multipotent, producing progeny that become distinct cell types. Clonally related cells can migrate rostrocaudally within domains that are restrained in the mediolateral and dorsoventral directions. A subset of rostrocaudal clones resemble those seen in the mammalian cerebral cortex, with respect to the crossing of functional boundaries, but all rostrocaudal clones differ from the cerebral cortical clones in the pattern of spread of sibling cells, with the rostrocaudal clones being more constrained in the mediolateral and dorsoventral directions. A role for lineage in the patterning of the chick forebrain is supported by these observations. In addition, these data suggest a role for cues within the telencephalic marginal zone that serve to guide clones in their rostrocaudal migration.