Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45

IRF-7 is the master regulator of type I interferon-dependent immune responses controlling both innate and adaptive immunity. Given the significance of IRF-7 in the induction of immune responses, many viruses have developed strategies to inhibit its activity to evade or antagonize host antiviral responses. We previously demonstrated that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu, F. X., King, S. M., Smith, E. J., Levy, D. E., and Yuan, Y. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5573-5578). In this report, we sought to reveal the mechanism underlying the ORF45-mediated inactivation of IRF-7. We found that ORF45 interacts with the inhibitory domain of IRF-7. The most striking feature in the IRF-7 inhibitory domain is two α-helices H3 and H4 that contain many hydrophobic residues and two β-sheets located between the helices that are also very hydrophobic. These hydrophobic subdomains mediate intramolecular interactions that keep the molecule in a closed (inactive) form. Mutagenesis studies confirm the contribution of the hydrophobic helices and sheets to the autoinhibition of IRF-7 in the absence of viral signal. The binding of ORF45 to the critical domain of IRF-7 leads to a hypothesis that ORF45 may maintain the IRF-7 molecule in the closed form and prevent it from being activated in response to viral infection.     

Linkage between energy status of perivascular cells and mineralization of the chick growth cartilage

The objective of this investigation was to investigate the relationship between the energy status of epiphyseal chondrocytes of the chick growth cartilage and the development of mineralization. A microfluorimetric scanning technique was used to measure the reduced pyridine nucleotide content of transverse sections of freeze-trapped cartilage; these measurements were related to tissue structure by scanning electron microscopy. The results of this study show that the energy status of cells in the hypertrophic region of the growth cartilage is more complex than was previously believed. In hypertrophic cartilage, most chondrocytes are in a reduced state. However, in the early hypertrophic region, the vascular channels that penetrate the cartilage from the metaphysis exert a profound local effect on the energy metabolism of perivascular chondrocytes. Thus, around each of the channels, there exists a zone of chondrocytes about 40-60 micron wide which exhibits a low fluorescence yield. The fluorescence level suggests that these perivascular cells have a higher level of oxidative metabolism than hypertrophic chondrocytes that are a distance (greater than 150 micron) from the vascular channels. This finding, in conjunction with our earlier observation that mineralization is first seen in the perivascular region, leads us to the conclusion that mineralization is associated with cellular oxidative activity. We now reject the long-held concept that in cartilage the development of mineralization is entirely due to tissue hypoxia.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.     

A new lace bug species of the genus Sinaldocader (Hemiptera: heteroptera, tingidae) from the turonian of southwestern Kazakhstan

Sinaldocader rasnitsyni sp.nov. (Heteroptera, Tingidae) is described from the Upper Cretaceous (Turonian) of southwestern Kazakhstan (Kzyl-Dzhar locality).     

Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Prostaglandin H synthetase (PGH synthetase) has been purified to homogeneity from sheep vesicular glands. The pure enzyme has a specific activity of about 40 microM of arachidonic acid consumed per minute per milligram of protein, which corresponds to a turnover number of 2800 min-1 per subunit. The purified enzyme was obtained by one-stage chromatography on DEAE-Toyopearl 650 from Tween 20-solubilized microsomes. A sensitive fluorometric assay for PGH synthetase activity using homovanillic acid (HVA) as electron donor has been proposed. It has been shown that homovanillic acid may be used as the electron donor and that in the presence of HVA the enzyme has an activity of approximately 40 microM/min/mg.     

Generation of lymphokine-activated killer cell activity from non-NK precursor cells

It is possible to generate high levels of lymphokine-activated killer (LAK) activity in short-term culture from cells enriched for natural killer (NK) activity. To determine whether LAK activity can also be generated from non-NK cells, we have depleted peripheral blood lymphocytes (PBL) of NK cells prior to culture with IL-2. NK activity in PBL is correlated with the intensity of staining with the lysosomotropic vital dye quinacrine. Quinacrine dim PBL, which are devoid of lytic NK cells, are capable of developing LAK activity following culture with IL-2. We have also separated PBL using the NK-associated NKH-1 marker. Depleting NKH-1+ cells eliminates NK activity but the ability to develop LAK activity is retained. NKH-1-depleted cells generate less LAK activity than unseparated or NKH-1-positive cells and do not proliferate as well as unseparated cells to IL-2. When NK-depleted cells are subsequently examined for the expression of the NKH-1 antigen, this marker is absent from most cells at Day 3 of IL-1 culture, but is expressed on an increasing number of cells by Days 6-8. These results suggest that LAK derived from non-NK cells is functionally and phenotypically similar to LAK from PBL-containing NK cells, and may be the result of the activation of an NK precursor population.     

Similarities between LAK cells derived from human thymocytes and peripheral blood lymphocytes: expression of the NKH-1 and CD3 antigens

Human thymocytes are devoid of NK cells but develop lymphokine-activated killer (LAK) activity after culture with recombinant interleukin-2 (rIL-2). The most active precursor for this activity appears to be a CD3-negative cell. The purpose of these studies was to compare the phenotype and functional activities of thymocyte and peripheral blood lymphocyte (PBL) LAK cells. Following culture, rIL-2-activated thymocytes resemble PBL-generated LAk and PBL NK cells. For each of these populations, lytic activity is highest in NKH-1-positive cells. Two-color fluorescence of each population also indicates that NKH-1+ cells are highly granular, as measured by staining with the lysosomotropic vital dye quinacrine. PBL, PBL-derived LAK cells, and thymus-derived LAK cells have a portion of cells that express both CD3 and NKH-1. However, approximately 60-80% of NKH-1+ cells lack detectable CD3. This suggests that both CD3+ and CD3- cells may be capable of LAK activity. Thymic-derived LAK cells respond to interferon in a manner very similar to NK and PBL-derived LAK cells, but lack the NK-associated CD16 antigen. Thus, despite the absence of NK cells in the thymus, it is possible to generate thymocyte LAK activity which bears a strong resemblance to LAK activity derived from peripheral blood lymphocytes.