Regulation of high-affinity leucine transport in escherichia coli

Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, low-affinity system (LIV-II). Expression of the high-affinity transport systems is altered by mutations in liv R and 1st R , genes for negatively acting regulatory elements, and by mutations in rho , the gene for transcription termination. All four genes for high-affinity leucine transport ( livJ, livK, livH , and livG ) are closely linked and have been cloned on a plasmid vector, pOX1. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that livJ and livK are separated by approximately one kilobase and give a gene order of livJ–livK–livH. livJ and livK appear to be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is inactivated by mutation or deletion. The results support the existence of separate promoters for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear to be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucinespecific transport systems by increasing the expression of livJ and livK , genes for the LIV-specific and leucine-specific binding proteins, respectively.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38209/1/400140410_ftp.pd

Hepatitis C Virus Controls Interferon Production through PKR Activation

Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection

Cloning and Characterization of the Genes Coding for the Membrane Components of the Leucine Transport in Escherichia Coli.

The high-affinity transport of the branched chain amino acids in E. coli is mediated by two binding protein dependent transport systems, LIV-I and LS, which are composed of multiple components. Genetic analyses have identified four complementation groups, livJ, livK, livH and livG which form a gene cluster at minute 74 on the E. coli chromosome. The livJ and livK genes code for LIV-BP and for LS-BP, respectively. Mutations in livH and livG have similar phenotypes which lack both LIV-I and LS transport activities. The liv genetic locus has recently been cloned into the pACYC184 plasmid vector to give the pOX1 plasmid. This thesis is concerned with the characterization of the livH and livG transport components. The genetic evidence indicating the presence of two genes has been confirmed by physically and functionally identifying the genes on the pOX1 plasmid using transposon mediated mapping, subcloning and DNA sequencing analysis. These experiments indicated that the livK, livH and livG genes form a polycistronic operon which appears to also contain an additional, previously unidentified gene, livM, located between the livH and livG genes. The livH, livM and livG genes have been subcloned into multicopy plasmid vectors and their products have been tentatively identified in vitro. The molecular weights of these components are 30,000, 27,000 and 22,000 respectively. All three proteins appear to be membrane-bound and are poorly expressed. The DNA sequence of the livH gene and its intergenic regions have been determined. The livH gene product is a rather basic protein containing several long hydrophobic segments which might be expected to transverse the cell membrane. An intergenic region of 47 bp exists between the livK and livH genes that contains a dyad symmetry of 7 GC base pairs which has the potential to form a stable stem-loop structure when transcribed. Finally livH-lacZ gene fusions containing the first ten amino acids of the livH protein have been constructed in vitro. The regulation of livH-lacZ gene expression has been studied and preliminary results indicate that the livH is under leucine control.Ph.D.BiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/160005/1/8412216.pd

Modulation of monocyte/macrophage-derived cytokine and chemokine profile by persistent Hepatitis C virus (HCV) infection leads to chronic inflammation

HCV infection presents a major public health problem, with more than 170 million people infected worldwide. Chronicity and persistence of infection constitute the hallmark of the disease. Although HCV is a hepatotropic virus, subsets of immune cells have been found to be permissive to infection and viral replication. Peripheral blood monocytes, attracted to the site of infection and differentiated into macrophages, and resident hepatic macrophages, known as Kupffer cells, are important mediators of innate immunity, through production of several chemokines and cytokines in addition to their phagocytic activity. HCV proteins have been shown to modulate the cytokine and chemokine production profile of monocytes/macrophages, as it is suggested by both in vitro and clinical studies. This modified expression profile appears crucial for the establishment of aberrant inflammation that leads to liver cirrhosis and hepatocellular carcinoma