DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

Thiocyanate transport in resting and IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels

SCN(-) (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN(-) is oxidized by H(2)O(2), a reaction catalyzed by lactoperoxidase, to produce OSCN(-) (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN(-) in the airway surface fluid, we studied transepithelial SCN(-) transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN(-) flux. cAMP and Ca(2+) regulatory pathways controlled SCN(-) transport through cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN(-)/Cl(-) exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN(-) transport. Measurements of H(2)O(2) production at the apical surface of bronchial cells indicated that the extent of SCN(-) transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN(-) transport mechanisms under resting and stimulated conditions. Defects in SCN(-) transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.     

Reorganization of prion protein membrane environment during low potassium-induced apoptosis in primary rat cerebellar neurons

We studied the changes occurring in the membrane environment of prion protein (PrP) during apoptosis induced by low potassium in primary rat cerebellar neurons. Ceramide levels increased during apoptosis-inducing treatment, being doubled with respect to time-matched controls after 24 h. Sphingomyelin levels were parallely decreased, while cholesterol and ganglioside contents were not affected. Changes in ceramide and sphingomyelin composition were exclusively restricted to a detergent-resistant membrane fraction. The pro-apoptotic treatment was accompanied by the down-regulation of PrP and of the non-receptor kinase Fyn. The levels of PrP and Fyn were correspondingly reduced in the detergent-resistant membrane fraction. In control cells, the membrane microenvironment separated by immunoprecipitation with anti-PrP antibody contained 80% of the detergent-resistant PrP and 35% and 38% of the sphingolipids and cholesterol respectively. Upon low potassium treatment, 20% of the PrP originally present in the detergent-resistant fraction was immunoprecipitated, together with 19% of sphingolipids and 22% of cholesterol. Thus, PrP in the immunoprecipitate from apoptotic cells was ninefold less than in control ones, while sphingolipids and cholesterol were about 50% with respect to controls cells. The molar ratio between cholesterol, sphingomyelin and ceramide was 15 : 6 : 1 in the PrP-rich environment from control neurons, and 6 : 2 : 1 in that from apoptotic cells.     

Inhibition of catalase activity as an early response of Arabidopsis thaliana cultured cells to the phytotoxin fusicoccin

In Arabidopsis thaliana cells, fusicoccin (FC) treatment induced an early and marked increase in the extracellular H(2)O(2) level. It also increased the huge hypo-osmotic stress-induced oxidative wave and, in addition, prevented the H(2)O(2) peak drop. These effects were apparently not linked to changes in either cytoplasmic pH or cytoplasmic free calcium concentration, since they occurred independently of the activity state of the plasma membrane (PM) H(+)-ATPase and neither influx nor efflux of (45)Ca(2+) was modified by FC. In the presence of diphenylene iodonium (DPI), inhibiting the PM NADPH oxidase presumably responsible for reactive oxygen species (ROS) production, no apoplastic H(2)O(2) development was detected either with or without FC. However, no increase in DPI-sensitive ferricyanide reduction, but rather a gradual decrease, occurred with FC. These results suggested that the H(2)O(2) increase observed with FC was not due to a overproduction of ROS but, more probably, to a reduced capability of FC-treated cells to degrade the H(2)O(2) formed. This view, at first supported by the finding that FC-treated cells failed to break down exogenously supplied H(2)O(2), was clearly confirmed by a series of measurements on exogenous catalase activity, tested in cell-free media of FC-treated samples. This assay, in fact, allowed ascertainment and partial characterization of an as yet unidentified factor increasingly accumulating in the incubation medium of FC-treated cells, behaving as a non-competitive catalase inhibitor and able to reduce markedly the cell's capability for H(2)O(2) scavenging.     

Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC(50)) and whether modulation of EC(50) affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20 degrees C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC(50); ssTnI-chimera Tn exchange induced a further decrease in EC(50) (in microM: endogenous Tn, 1.35 +/- 0.08; cTnI, 1.04 +/- 0.13; ssTnI-chimera Tn, 0.47 +/- 0.03). EC(50) was also decreased by application of 100 microM bepridil (control: 2.04 +/- 0.03 microM; bepridil 1.35 +/- 0.03 microM). Maximum tension was not different between any groups. Despite marked alterations in EC(50), none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca(2+) sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC(50). This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.     

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.     

Automated genome-wide visual profiling of cellular proteins involved in HIV infection

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.     

Precisely timed oculomotor and parietal EEG activity in perceptual switching

Blinks and saccades cause transient interruptions of visual input. To investigate how such effects influence our perceptual state, we analyzed the time courses of blink and saccade rates in relation to perceptual switching in the Necker cube. Both time courses of blink and saccade rates showed peaks at different moments along the switching process. A peak in blinking rate appeared 1,000 ms prior to the switching responses. Blinks occurring around this peak were associated with subsequent switching to the preferred interpretation of the Necker cube. Saccade rates showed a peak 150 ms prior to the switching response. The direction of saccades around this peak was predictive of the perceived orientation of the Necker cube afterwards. Peak blinks were followed and peak saccades were preceded by transient parietal theta band activity indicating the changing of the perceptual interpretation. Precisely-timed blinks, therefore, can initiate perceptual switching, and precisely-timed saccades can facilitate an ongoing change of interpretation.