The thymidylate synthase (TS) gene, which is induced at the G(1)-S transition in growth-stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP-1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G(1)-S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant-negative LSF (LSFdn) prevents the increase in TS enzyme levels during G(1)-S, and induces apoptosis in growth- stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5-fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S-phase by targeting a single gene that is critical for cell survival.
05). PA-specific gamma interferon (IFN-␥) and interleukin-4 (IL-4) CD4؉ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.
Matrix metalloproteinases (MMPs), a class of enzymes responsible for the degradation of extracellular matrix proteins, play important roles in inflammatory and immune responses. In skin, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are normally inactive but can be expressed during tissue injury. Both degrade collagen IV and other critical components of the basement membrane zone that separates the epidermis from the dermis. The expression of MMP-2 and -9 was studied in sulfur mustard (SM)-exposed ear skin from mice to determine their role in tissue vesicant injury. Punch biopsies of mouse ears were collected between 6 and 168 h after exposure to 97.5 mM (0.08 mg) SM diluted in CH(2)Cl(2). They were examined histologically and assayed for MMP-2 and -9 expression by gelatinase activity assays, real-time reverse transcriptase-polymerase chain reaction and Western blot analysis. A time-related increase in overall gelatinase activity was observed in SM-treated ears. At 168 h after SM exposure, the relative levels of MMP-9 mRNA were increased 27-fold and MMP-9 protein 9-fold when compared with the control (CH(2)Cl(2) treated) ears. In contrast, there were no observable increases in the MMP-2 mRNA or protein levels between treated and control ears. These observations suggest the differential expression of MMP-2 and -9 during the cutaneous response to SM injury and suggest a role for MMP-9 in SM-induced injury.
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