Expression of pro form of prostate-specific antigen by mammalian cells and its conversion to mature, active form by human kallikrein 2

To study the expression, biosynthesis, and processing of prostate-specific antigen (PSA) in mammalian cells, recombinant PSA was expressed in Syrian hamster tumor cell line AV12-664 (AV12-PSA). Expression of PSA was monitored by the Tandem-MP PSA assay. PSA was secreted into the medium during the logarithmic phase of cell growth at >9 microg/ml and was stable. The PSA purified from spent medium of AV12-PSA cells did not exhibit any enzymatic activity and did not complex with the protease inhibitor, alpha-1-antichymotrypsin. These findings indicated that an inactive form of PSA was expressed by AV12-PSA cells. NH2-terminal sequencing confirmed the identity of the PSA purified from the spent medium of AV12-PSA cells to be pro-PSA. This demonstrates that PSA is expressed as pro-PSA by mammalian cells and suggests that pro-PSA may be present in biological fluids. Human kallikrein 2 (hK2), another member of the hK family, is also expressed predominantly in prostate epithelium. Although hK2 has been shown to exhibit trypsin-like activity, little is known about its natural substrates. Using purified proteins, we show that hK2 can convert pro-PSA to mature, enzymatically active PSA, thus establishing a physiological connection between hK2 and PSA. These findings imply that hK2 may be regulating PSA activity in vivo.     

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.     

The N-terminal domain of human GABA receptor rho1 subunits contains signals for homooligomeric and heterooligomeric interaction

gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.     

Induction of telomerase activity by UV-irradiation in Chinese hamster cells

Telomerase is a ribonucleoprotein whose activity has been detected in germline cells and in neoplastic and immortal cells. Telomerase compensates the telomere loss arising by the end replication problem by synthesizing telomeric repeats at the 3' end of the eukaryotic chromosomes. Telomerase is reactivated during cancer progression in human and mice. In order to determine whether the telomerase activity can be upregulated in vitro in response to DNA damaging agents, we examined the telomerase activity in five Chinese hamster cell lines following exposure to 5 J/m2 or 40 J/m2 UV-C radiation. All the cell lines tested showed an increase in telomerase activity in the PCR-based telomeric repeat amplification protocol (TRAP) in a dose dependent manner. This increase in telomerase activity correlated well with the number of cells being in the S and G2/M phase after UV exposure. However, in unirradiated control cells, similar levels of telomerase activity were observed in different phases of the cell cycle. Furthermore, telomeric signals were clustered in one or more parts of the disintegrating nuclear particles of the apoptotic cell as detected by fluorescence in situ hybridization (FISH). This is the first study to demonstrate the induction of telomerase activity following exposure to DNA-damaging agents like UV radiation in Chinese hamster cells in vitro.     

Pulmonary surfactant protein SP-B interacts similarly with dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine in phosphatidylcholine/phosphatidylglycerol mixtures

Porcine pulmonary surfactant-associated protein SP-B was incorporated into bilayers of chain-perdeuterated dipalmitoylphosphatidylglycerol (DPPG-d62) and into bilayers containing 70 mol % dipalmitoylphosphatidylcholine (DPPC) and 30 mol % DPPG-d62 or 70 mol % chain-perdeuterated DPPC (DPPC-d62) and 30 mol % DPPG. The effect of SP-B on the phase behavior, lipid chain order, and dynamics in these bilayers was examined using deuterium nuclear magnetic resonance (2H-NMR). In both DPPG-d62 and the mixed lipid system, SP-B is found to have little effect on chain order in the liquid crystalline phase. With 11% (w/w) SP-B present, both bilayer systems display a continuous change from liquid crystal to gel with no evidence of two-phase coexistence near the transition. Despite its limited effect on chain order in these bilayers, SP-B is found to strongly perturb chain deuteron transverse relaxation in the liquid crystal and gel phases of DPPG-d62 and the DPPC/DPPG (7:3) mixtures. The observation that SP-B associates with the bilayer in a way which substantially alters the slow motions responsible for transverse relaxation without significantly affecting chain order in either the liquid crystal or gel phases may place some constraints on possible models for that association.     

Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients

Lymphoreticular malignancies, collectively called posttransplant lymphoproliferative disorders (PTLD), eventually develop in 2-5% of organ transplant recipients. They frequently undergo regression when immunosuppression is reduced or stopped. This feature has been associated with a previous or de novo Epstein-Barr virus (EBV) infection. We herein describe immunotherapy with autologous lymphokine-activated killer (LAK) cells in seven patients with PTLD (four EBV-positive patients and three EBV-negative patients). Autologous peripheral blood mononuclear cells were obtained by leukapheresis, depleted of monocytes, and cultured in the presence of interleukin 2 for 10 to 11 days. A single dose of 5.2 x 10(9) to 5.6 x 10(10) LAK cells was given intravenously. Systemic interleukin 2 was not administered. The four patients with EBV+ PTLD had complete tumor regression; two of them developed controllable rejection. Three patients are well 13-16 months after treatment; the fourth patient died of pneumonia 41 days after infusion. Three patients with EBV- lymphomas had no response despite prior evidence that their tumors also were subject to immune surveillance. Two of these three patients died after being given other treatment, and the third patient has persistent tumor. In conclusion, autologous LAK cell infusion was effective for treatment of four EBV+ organ transplant recipients. LAK cell efficacy for three patients with EBV- PTLD was not evaluable under the management circumstances in which this treatment was utilized.