The SasS -SasR two-component signal transduction system required for early Myxococcus xanthus multicellular *development

Initiation of Myxococcus xanthus multicellular development requires both nutrient limitation and high cell density. The extracellular signal, A signal, which consists of a set of amino acids at specific concentrations, serves as a cell density signal in M. xanthus early development. A reporter gene, designated 4521, that requires both starvation and A signal for developmental expression was used to identify mutations in the signal transduction pathways. A group of point mutations located in the chromosomal sasB locus that bypasses both requirements was previously isolated. One of these point mutations, sasB7, was mapped to the sasS gene, which is predicted to encode a transmembrane histidine protein kinase required for normal development. SasS is a positive regulator of 4521 and a candidate A signal sensor. This dissertation continues the characterization of the sasB locus, focusing on the sasR gene and the functional relationship of SasS and SasR. ^ The sasR gene is located 2.2-kb downstream of sasS. It is predicted to encode an NtrC-like response regulator, which belongs to the family of sigma54 transcriptional activators. SasR is a positive regulator of 4521 gene and is required for normal development. The sasR mutant displays phenotypes similar to that of sasS mutant. Both SasS and SasR are required for the A-signal-dependent 4521 expression. Genetic epistasis analysis indicates that SasR functions downstream of SasS. Biochemical studies show that SasS has autokinase activity, and phosphorylated SasS is able to transfer its phosphate to SasR. We propose that SasS and SasR form a two-component signal transduction system in the A signal transduction pathway. ^ To search for the genes regulated by SasS and SasR, expression patterns of a group of developmental genes were compared in wild-type and sasS null mutant backgrounds. SasS and SasR were found to positively regulate sasN and 4521. The sasN gene was previously identified as a negative regulator of 4521, located at about 170-bp downstream of sasR. It is required for normal fruiting body development. Based on the above data, a regulatory network consisting of sasS, sasR, sasN, and 4521 is hypothesized, and the interactions of the components in this network can now be further studied. ^

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Cross -packing among retroviruses and characterization of the feline immunodeficiency virus (FIV) packaging signal: Implications for the development of FIV-based gene transfer systems

Feline immunodeficiency virus (FIV)-based gene transfer systems are being seriously considered for human gene therapy as an alternative to vectors based on primate lentiviruses, a genetically complex group of retroviruses capable of infecting non-dividing cells. The greater phylogenetic distance between the feline and primate lentiviruses is thought to reduce chances of the generation of recombinant viruses. However, safety of FIV-based vector systems has not been tested experimentally. Since primate lentiviruses such as human and simian immunodeficiency viruses (HIV/SIV) can cross-package each other's genomes, we tested this trait with respect to FIV. Unexpectedly, both feline and primate lentiviruses were reciprocally able to both cross-package and propagate each other's RNA genomes. This was largely due to the recognition of viral packaging signals by the heterologous proteins. However, a simple retrovirus such as Mason-Pfizer monkey virus (MPMV) was unable to package FIV RNA. Interestingly, FIV could package MPMV RNA, but not propagate it for further steps of replication. These findings suggest that upon co-infection of the same host, cross-packaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential. ^ In order to understand the packaging determinants in FIV, we conducted a detailed mutational analysis of the region thought to contain FIV packaging signal. We show that the first 90–120 nt of the 5′ untranslated region (UTR) and the first 90 nt of gag were simultaneously required for efficient FIV RNA packaging. These results suggest that the primary FIV packaging signal is multipartite and discontinuous, composed of two core elements separated by 150 nt of the 5 ′UTR. ^ The above studies are being used towards the development of safer FIV-based self-inactivating (SIN) vectors. These vectors are being designed to eliminate the ability of FIV transfer vector RNAs to be mobilized by primate lentiviral proteins that may be present in the target cells. Preliminary test of the first generation of these vectors has revealed that they are incapable of being propagated by feline proteins. The inability of FIV transfer vectors to express packageable vector RNA after integration should greatly increase the safety of FIV vectors for human gene therapy. ^