Uninhibited Cells Research Articles

The role of ROS levels on the activation of SMAD2 by TGFβ1 in corneal fibroblasts

AbstractPurposesTGF‐1/TGFβR‐induced corneal myofibroblast genesis is mediated by the positive feedback loop (TGFβR ‐>> p‐SMAD2↑ ‐>> ROS↑‐>>TRPV1↑ ‐>> Ca↑‐>> p‐p38↑‐>> p‐SMAD2↑‐>ROS..‐> p‐SMAD2↑). Inhibition of pp38, ROS, TRPV1, Ca influx or N‐acetyl cysteine (NAC; 1 µM), a ROS scavenger, reduces SMAD2 phosphorylation (PLOS1, 2013: e77300). Our studies seek to identify signal transduction components of the loop.Methods4th generation cultures of fibroblasts derived from donor cadaver corneas (HCF) seeded in a FBS‐free fibroblast medium for 24h were incubated with inhibitors (15 min at 10‐50 x of inhibitor Kd) and the changes in p‐SMAD2 and p‐p38 upon addition of TGFβ1 (2 ng/ml for 5‐60 min) were determined by Western blot. Untreated HFCs showed nil p‐SMAD2 and a low p‐p38 level.ResultsAddition of TGFβ1 to uninhibited cells caused a 4.7‐fold p‐p38 increase (n=3) and the de novo accumulation of p‐SMAD2. Inhibition of maternal embryonic leucine zipper kinase (MELK) by 100 nM OTSSP168, phospholipase D by 1 µM FIPI, PI3Kα by 50 nM PI3K Inhibitor #2, or the superoxide conversion to H2O2 in the mitochondria by 500 µM Mito‐Tempo resulted in > 5‐fold increase in p‐p38 and pronounced or full inhibition of the de novo SMAD2 activation by TGFβ1. The increase in p‐p38 and inhibition of SMAD2 activation was also elicited by incubation with the cellular oxidant p‐Nitrophenyldisulfide (NPDS; 10 nM). Next, we treated the cells with NAC, 0‐500 nM NPDS and TGFβ. As the [NPDS] was increased, the inhibitory effect of NAC on both [p‐p38] and [p‐SMAD2] was decreased and canceled at [NPDS] = 15 nM. Further increases in [NPDS] thought, elevated [p‐p38] but inhibited SMAD2 activation by TGFβ1.ConclusionFibroblast activation by TGFβ1/TGFβR requires a very precise ROS level. The inhibitory effect of some signal transduction pathways may occur indirectly through their effect of the redox cellular status. Inflammations or infections that increases ROS may delay the wound healing response.

S199 Generation of healthy and pathological synchronous events in the hippocampus: Modulation of cellular and network parameters results in altered dynamics

Cortical activity is characterized by behavior associated alternation of network states, such as physiological sharp-wave ripples (SWRs) and theta phase modulated gamma in the hippocampus. Epileptiform bursts are characteristic in the pathological case. In in vitro mouse hippocampal slice preparation the activity can be switched among SWR, gamma and epileptic states. We asked: – How are SWRs initiated and what is the difference between in vitro SWRs and epileptiform events? – How does the same network generates different dynamics? SWRs and epileptiform bursts share similar initiation mechanisms, and express high-frequency oscillations (HFOs) with different features. Both during SWRs and epileptiform events activity is stochastically initiated in the recurrent excitatory network of CA3 pyramidal cells. As activity increases PV+ inhibitory neurons are recruited. During SWRs the HFO is generated in the reciprocally-connected network of inhibitory PVBCs, while in the pathological case, synchronous failure of perisomatic inhibition triggers massive pyramidal cell burst firing. The physiological ripple oscillation is the result of phasic perisomatic inhibitory currents, pathological HFOs are predominantly population spikes of partially synchronous, massively bursting, uninhibited pyramidal cells. The two cases can be distinguished by analyzing the HFO spectrum and the correlation of signals among nearby electrodes. Two basic parameters: neuronal excitability and the strength of synaptic connections will influence the dynamics of the network. SWRs, gamma oscillations and epileptiform events can morph into each other upon manipulating the above parameters. The gamma generating state and the epileptic state are very close to each other in the parameter space. Support: OTKA K83251 , K115441 , NNF85659 , ERC 2011-ADG-294313 , and the HBP .

β-catenin and PI3Kδ inhibition expands precursor Th17 cells with heightened stemness and antitumor activity.

ICOS costimulation generates Th17 cells with durable memory responses to tumor. Herein, we found that ICOS induces PI3K/p110δ/Akt and Wnt/β-catenin pathways in Th17 cells. Coinhibiting PI3Kδ and β-catenin altered the biological fate of Th17 cells. Th17 cells inhibited of both pathways expressed less RORγt, which, in turn, reduced their ability to secrete IL-17. Unexpectedly, these cells were more effective (than uninhibited cells) at regressing tumor when infused into mice, leading to long-term curative responses. PI3Kδ inhibition expanded precursor Th17 cells with a central memory phenotype that expressed nominal regulatory properties (low FoxP3), while β-catenin inhibition enhanced Th17 multifunctionality in vivo. Remarkably, upon TCR restimulation, RORγt and IL-17 rebounded in Th17 cells treated with PI3Kδ and β-catenin inhibitors. Moreover, these cells regained β-catenin, Tcf7, and Akt expression, licensing them to secrete heightened IL-2, persist, and eradicate solid tumors without help from endogenous NK and CD8 T cells. This finding shines a light on ways to repurpose FDA-approved drugs to augment T cell-based cancer immunotherapies.

Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50-300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.

Tunica Arterial Adventitia: A New Exploration in Intimal Hyperplasia

Vascular interventions have become widely adopted for treatment of coronary and peripheral arterial atherosclerosis. Although there is increasing utilization of the new technology, restenosis still retarded the long-term outcome of the intervention. Histological studies have revealed that uninhibited neointimal cells play a vital role in the post-injury response. Among them, the majority of neointimal cell express smooth muscle cell (SMC) markers and thus, it was believed that the SMC in neighborhood migrate into the subintimal space, proliferate and secrete extracellular matrix, therefore contributing to the intimal hyperplasia. Many studies in this thematic review focus on the specifics of the injury, the cytokines and chemokines that drive SMC, and the nature of the migrated SMC. In addition, more studies documented that adventitia is an active participator instead of bystander through geneticlabeling and tracking adventitia in animal model. This brief review focuses on the recent findings of adventitia in vascular response after injury and highlights that multiplineage cells in adventitia contribute to intimal hyperplasia synergistically with SMC.

ZMIZ1 Collaborates with NOTCH1 During Induction and Maintenance of T-Cell Acute Lymphoblastic Leukemia

Abstract 75Activating NOTCH1 mutations are found in 40–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, most leukemia-associated NOTCH1 mutations fail to induce leukemia. This observation suggests that cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. In murine retroviral/transposon insertional mutagenesis screens, induction of the Zmiz1 gene was frequently associated with activation of the Notch1 receptor during leukemogenesis (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT-like family that has been implicated for prostate cancer survival. It directly interacts with the androgen receptor to enhance its transcriptional activity. To investigate the role of ZMIZ1 during leukemogenesis, activating NOTCH1 mutations and ZMIZ1 were transduced into hematopoietic progenitor cells. These cells were then used to reconstitute lethally irradiated mice. ZMIZ1 or NOTCH1 alone failed to induce T-ALL after 1 year of observation. In contrast, ZMIZ1 and NOTCH1 in combination induced T-ALL with ∼50% penetrance by 100 days after transplantation. These data show that ZMIZ1 can promote leukemogenesis in cooperation with NOTCH1. To determine the relevance of ZMIZ1 to human leukemia, we screened 15 primary human adult T-ALL samples for ZMIZ1 mRNA and protein. 20% expressed ZMIZ1. In publically available data sets, ZMIZ1 gene expression was significantly enriched by ∼2-fold in early thymocyte precursor ALL (ETP-ALL) samples. The ETP-ALL subgroup comprises about 13% of all T-ALL and may have a highly unfavorable prognosis. We discovered expression of ZMIZ1 in two ETP-like cell lines. To investigate whether ZMIZ1 is a potential therapeutic target, we transduced these cell lines with shRNA directed against ZMIZ1. ZMIZ1 inhibition reduced cell size, increased apoptosis by ∼2-fold, and reduced growth by 75–94%. Furthermore, ZMIZ1 knockdown overcame resistance to NOTCH signaling blockade with g-secretase inhibitors. Since the glucocorticoid receptor is highly homologous to the androgen receptor, we considered the possibility that ZMIZ1 inhibition may promote glucocorticoid resistance. However, we treated ZMIZ1-inhibited T-ALL cell lines with increasing doses of dexamethasone. ZMIZ1-inhibited T-ALL cell lines were twice as sensitive to dexamethasone than uninhibited cells. These data suggest that ZMIZ1 is required for leukemia growth and survival. Inhibition of ZMIZ1 may potentially enhance targeting of T-ALL with NOTCH pathway inhibitors and glucocorticoids. To determine the mechanism underlying ZMIZ1 function, we performed gene expression profiling. We identified C-MYC as a potential downstream target of ZMIZ1. C-MYC is also a direct target of NOTCH1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous C-MYC expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by ∼7000 fold, induced C-MYC target gene expression, and rescued the C-MYC dependent cell line. ZMIZ1 inhibition lowered C-MYC levels by ∼93%. The interaction between ZMIZ1 and NOTCH appeared to be specific for C-MYC, as modulation of ZMIZ1 levels did not affect the NOTCH1 target genes Hes1, Dtx1, and Cd25. Downregulation of C-MYC partly phenocopied the effects of ZMIZ1 downregulation. However, ectopic expression of C-MYC failed to rescue the growth of ZMIZ1-inhibited cells. These data suggest that C-MYC is an essential but insufficient downstream effector of ZMIZ1 function. In conclusion, ZMIZ1 is new potential therapeutic target in a subset of T-ALL. It functionally interacts with NOTCH1 to promote C-MYC expression and activity. Disclosures:No relevant conflicts of interest to declare.

Loss of Inducible Nitric Oxide Synthase Expression in the Mouse Renal Cell Carcinoma Cell Line RENCA Is Mediated by MicroRNA miR-146a

Tumor-associated macrophages can potentially kill tumor cells via the high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS); however, tumor-associated macrophages actually support tumor growth, as they are skewed toward M2 activation, which is characterized by low amounts of NO production and is proangiogenic. We show that the mouse renal cell carcinoma cell line, RENCA, which, on stimulation, expresses high levels of iNOS mRNA, loses its ability to express the iNOS protein. This effect is mediated by the microRNA miR-146a, as inhibition of RENCA cells with anti-miR- 146a restores iNOS expression and NO production (4.8 ± 0.4 versus 0.3 ± 0.1 μmol/L in uninhibited cells, P < 0.001). In vivo, RENCA tumor cells do not stain for iNOS, while infiltrating tumor-associated macrophages showed intense staining, and both cell types expressed iNOS mRNA. Restoring iNOS protein expression in RENCA cells using anti-miR-146a increases macrophage-induced death of RENCA cells by 73% (P < 0.01) in vitro and prevents tumor growth in vivo. These results suggest that, in addition to NO production by macrophages, tumor cells must produce NO to induce their own deaths, and some tumor cells may use miR-146a to reduce or abolish endogenous NO production to escape macrophage-mediated cell death. Thus, inhibiting miR-146a may render these tumor cells susceptible to therapeutic strategies, such as adoptive transfer of M1-activated macrophages.

A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux

Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average influx was 33.9±6.4fmolcm−2s−1). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50–150fmolcm−2s−1 range. Uptake was inhibited using threo-β-benzyloxyaspartate, and average surface flux in replicate cells (1.1±7.4fmolcm−2s−1) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions.

A key role for dense granule secretion in potentiation of the Ca 2+ signal arising from store-operated calcium entry in human platelets

Recent work has demonstrated a role for Na +/Ca 2+ exchange in potentiation of the Ca 2+ entry elicited through the human platelet store-operated channel by controlling a Mn 2+-impermeable Ca 2+ entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na +/Ca 2+ exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn 2+-impermeable Ca 2+ entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca 2+ signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca 2+ signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.

Kdo2‐Lipid A stimulated GIVA Phospholipase A2 catalyzed AA release is Phosphatidic Acid Phosphohydrolase‐1 Dependent

Macrophages respond to toll‐like receptor 4 (TLR‐4) agonists, such as LPS and Kdo2‐Lipid A, by upregulating cyclooxygenase 2 (COX‐2) and releasing prostaglandins. Group IVA phospholipase A2 (GIVA PLA2) catalyzes the deacylation of membrane‐bound arachidonic acid (AA) from the sn‐2 position of glycerophospholipids, which can then be further metabolized by COX‐2 in the synthesis of prostaglandins. Phosphatidic acid phosphohydrolase‐1 (PAP‐1) is a Mg+2 dependent, membrane‐associated protein involved in the synthesis of triacylglycerol. We show herein that GIVA PLA2‐mediated AA release and COX‐2 expression through TLR‐4 signaling mechanism requires PAP‐1. PAP‐1 chemical inhibition decreased COX‐2 mRNA transcript production, COX‐2 protein expression and AA release from macrophages. Addition of diacylglycerol, the end‐product of PAP‐1 catalysis, enhanced AA release from uninhibited cells and restored the AA release in inhibited cells. TLR‐4 activation increased the levels of endogenous cellular DAG within two minutes of stimulation and this increase was sensitive to PAP‐1 inhibition. Furthermore, PA phosphohydrolase activity assays showed that PAP‐1 activity translocated from the cytosolic fraction to the membrane fraction within two minutes of stimulation. DAG add‐back experiments demonstrate that TLR‐4 induced COX‐2 expression is enhanced by the addition of exogenous DAG. Additionally, PAP‐1 chemical inhibition reduced the synergistic release of AA from cells stimulated with TLR‐4 agonist and ATP, while not affecting the magnitude of synergistic activation.

Cyclic Pressure Stimulates DNA Synthesis through the PI3K/Akt Signaling Pathway in Rat Bladder Smooth Muscle Cells

Previous studies demonstrated that the bladder exhibited severe tissue remodeling following spinal cord injury. In such pathological bladders, uninhibited non-voiding contractions subject bladder cells to cyclic oscillations of intravesical pressure. We hypothesize that cyclic pressure is a potential trigger for tissue remodeling in overactive bladder. Using a custom-made setup, rat bladder smooth muscle cells (SMC) in vitro were exposed to cyclic hydrostatic pressure (40 cm H2O) at either 0.1 Hz or 0.02 Hz frequency for up to 24 h. When compared to static control and cells exposed to 0.02-Hz cyclic pressure, SMC exposed to 0.1-Hz cyclic pressure contained significantly (p < 0.05) higher amounts of DNA. We confirmed that the increase in DNA was due to increased cell proliferation, indicated by increased BrdU incorporation, but not due to decreased apoptosis rates in response to cyclic pressure. In addition, significant (p < 0.05) elevation of Akt phosphorylation in SMC following exposure to cyclic pressure and lack of pressure-induced SMC hyperplasia in the presence of PI3K inhibitors, wortmannin and LY294002, indicated the involvement of the PI3K/Akt pathway in the proliferative response of SMC to cyclic pressure. We concluded that chronic exposure to intravesical pressure oscillation may be a potential trigger for bladder tissue remodeling.

Resistance to EGFR kinase inhibitor PD158780 in head and neck cancer cell lines

Problem: Overexpression of the epidermal growth factor receptor (EGFR) is common in head and neck squamous cell carcinoma (HNSCC) and is believed to contribute significantly to malignant behavior in this disease. However, EGFR kinase inhibitors (EKIs) have been less successful than predicted at impeding the growth of these tumors. To begin to understand the mechanism(s) of resistance to these agents, the effect of the EKI PD158780 on EGFR-activated signaling molecules was assessed in vitro. Methods: The effect of PD158780 on basal and EGF-stimulated protein phosphorylation and proliferation were assessed in 3 HNSCC cell lines using immunoblot and colorimetric cell density assays, respectively. Results: All 3 cell lines were shown to exhibit constitutive EGFR, Shc, and Erk phosphorylation in the absence of exogenous stimulation. PD158780 completely eliminated constitutive EGFR phosphorylation at low concentrations; higher concentrations were necessary to eliminate basal Shc and Erk phosphorylation. In EGF-stimulated cells, elimination of EGFR phosphorylation required 100-fold higher concentrations of PD158780, and Erk activation persisted, even with high-dose inhibitor and in the absence of detectable EGFR phosphorylation. Other growth factors that had little effect on Erk activity in uninhibited cells could stimulate significant increases in Erk phosphorylation in the presence of PD158780. This correlated with an increased ability of these ligands to stimulate proliferation in PD158780-treated cells. Conclusion: The preservation of Erk activity despite dramatic reduction or elimination of EGFR phosphorylation represents a potentially significant pathway to EKI resistance in HNSCC. Measurement of EGFR phosphorylation as an endpoint is clearly inadequate to assess the success of these agents, and other growth factor systems may figure prominently in the persistence of the EGFR-inhibited tumors. Significance: Incomplete elimination of downstream proliferative signals and/or enhanced response to alternate growth factors may explain resistance to EKIs. Thus, combination with other inhibitory agents may be necessary to achieve optimal success. Support: American Head & Neck Society 2003 Pilot Research Grant (MJJ)

Flow cytometry in the study of mitochondrial respiratory chain disorders

We have developed a flow cytometric assay to measure the oxidative capacity of cultured lymphoblasts as a possible screening test for patients suspected of having a defect of the mitochondrial respiratory chain. Cells were incubated overnight in serum free media, followed by incubation with dihydroethidium with and without rotenone, and then analysed using flow cytometry to measure fluorescence. Inhibition with rotenone gave an increase in fluorescence compared to uninhibited cells. The change in fluorescence was significantly lower in four of the six patient cell lines, with a correlation between the activity of complex I and change in fluorescence. This method may be applicable to cell lines with defects in other complexes of the respiratory chain.

Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells

Loss of long-term hematopoietic stem cell function in vitro is associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine Sca-1(+)c-kit(+)Lin(-) cells were cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating factor (G-CSF), stem cell factor, flk-2 ligand, and thrombopoietin for 7 days. Cells amplified 2000-fold were then stained with Hoechst 33342, separated into G(0)/G(1) (72% +/- 3%) or S/G(2)/M (27% +/- 3%) fractions by flow sorting, and injected into lethally irradiated mice. Although long-term (more than 6 months) engraftment of lymphoid and myeloid lineages was greater in primary and secondary recipients of expanded cells residing in G(0)/G(1) at the time of transplantation, there were no noted differences in the short-term (less than 6 weeks) recovery kinetics of circulating blood cells. When hematopoietic cells were expanded in cultures containing the tetrapeptide stem cell inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor cycling prior to transplantation, again there were no differences observed in short-term reconstitution by inhibited or uninhibited cells. Interestingly, AcSDKP significantly accelerated engraftment by expanded hematopoietic cells when administered in vivo at the time of transplantation. Leukocytes recovered to 20% of normal levels approximately 1 week faster, and thrombocytopenia was largely abrogated in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can accelerate the engraftment of ex vivo expanded hematopoietic progenitors, which suggests a relatively simple approach to improve their clinical utility, its effects appear unrelated to cell cycle arrest. (Blood. 2000;95:2829-2837)

Cysteine derivatives protect against UV-induced reactive intermediates in human keratinocytes: the role of glutathione synthesis.

Several recent studies have shown cysteine derivatives can protect against negative effects of UV exposure. In this study, an attempt was made to correlate cellular bioavailability and metabolism of cysteine derivatives with protection against UV-induced reactive intermediates. Human keratinocytes were treated with cysteine, N-acetylcysteine (NAC), cysteine-ethylester (CYSET) and N-acetylcysteine-ethylester. The uptake of the compounds and their metabolism to cysteine and eventually to glutathione (GSH) was measured. Large differences in uptake were observed, with CYSET resulting in the highest and NAC in the lowest intracellular thiol levels. The increase in intracellular GSH was similar for all derivatives with a maximum of 23-54% over the control level. Protective efficacy of the derivatives was measured as the inhibition of binding of UV-induced reactive intermediates from 8-methoxypsoralen. There was only a small difference between the compounds, with maximum protection of 25-31%. No relation was found between total intracellular thiol and protection. However, for NAC, there was a linear relation between GSH level and protective efficacy (r = 0.94). Even though this was not clear for the other derivatives (r = 0.55 for CYS; r = 0.60 for CYSET; r = 0.70 for NACET), it indicates that GSH synthesis is an important factor. This was confirmed by experiments using cells with irreversibly inhibited GSH synthesis. Even though the total intracellular thiol level was comparable to uninhibited cells, protection was decreased. We conclude that the intracellular GSH increase is the most important factor in photoprotection by cysteine derivatives.

Conformational Aberrance of the Sendai Virus Fo Protein in Thapsigargin-Treated Cells Allowing Exiot from the Endoplasm Reticulum but Causing Arrest at the Golgi Complex1

Thapsigargin and ionomycin inhibited the intracellular transport of the Sendai virus F0 protein. Depletion of Ca2+ from intracellular Ca2+ store(s) is critical for the inhibition, since ionomycin was more effective in the absence of extracellular Ca2+ than in its presence. Transport of F0 was arrested between the trans-Golgi complex and the plasma membrane [Ono, A. and Kawakita, M. (1994) J. Biochem. 116, 649-656], but only when thapsigargin was added before the synthesis of F0. This implies that F0 was committed to later arrest in the endoplasmic reticulum. Non-reducing SDS-polyacrylamide gel electrophoresis revealed a conformational abnormality of F0 immediately after pulse-labeling in thapsigargin-treated cells. The abnormality did not affect the exit of F0 from the endoplasmic reticulum, but paralleled its later arrest at the trans-Golgi stage. Pulse-labeled and 1-h-chased F0 was endoglycosidase H-resistant even in thapsigargin-treated cells, but was not recognized by mAb f-49, a monoclonal antibody that recognizes the corresponding F0 intermediate in uninhibited cells. The misfolded F0 may escape from recognition by means of the quality control system of the endoplasmic reticulum, but another system in the Golgi complex may complement the former. The arrested F0 was rapidly degraded.

Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination.

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p-beta-galactosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post-ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the 'run-off' polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.

Synthetic inhibitors of HIV-1 protease inhibit virus maturation and infectivity of virions

The biochemical mechanism of lymphocyte dysfunction with adenosine deaminase deficiency has been investigated using cultured phytohemagglutinin stimulated normal peripheral blood lymphocytes and the adenosine deaminase (ADA) inhibitor 2′-deoxycoformycin. The addition of deoxyadenosine to ADA-inhibited (but not to uninhibited) cells generated increased dATP pools (up to 50-fold greater than controls) and depressed the mitogen response. dATP Accumulation was accompanied by depletion of the other three deoxynucleoside triphosphate (dNTP) pools (dTTP, dCTP, and dGTP). Suppression of the mitogen response could be prevented (“reversed”) to 90% of control levels by the addition of deoxynucleoside precursors for the depleted dNTPs at the initiation of mitogen stimulation. “Reversal” restored the dTTP and possibly the dGTP pools. Thus the mechanism of toxicity in this model appears to be inhibition of ribonucleotide reductase by massive accumulation of dATP, resulting in starvation for the other three deoxyribonucleoside triphosphates. “Reversibility” of this toxicity by providing sources for the missing three deoxynucleoside triphosphates argues for ribonucleotide reductase inhibition rather than other mechanisms of deoxyadenosine toxicity in this model.

Reconstitution of primary mixed-lymphocyte culture reactions inhibited with class I antibodies by a product of histoincompatible spleen cells

Primary MLC reactions with spleen cells from mice of conventional inbred or congenic origin and incompatible at the entire H-2 or at K- or D-regions could be inhibited by pretreating stimulator but not responder cells with mAb to Class I MHC antigens. Inhibited MLC reactions, initiated in the presence of a product (SEPIR) present in supernatants after short-term cultivations of one-way histoincompatible spleen cells of the same haplotype as MLC cells, could be restored specifically to normal activities. It is suggested that the reconstituting factor represents a product of Class I antigen recognition capable of compensating the prevented recognition of these antigens by MLC responder cells. SEPIR was ineffective in restoring Class II antigen-inhibited MLC responses. Because the product might be formed spontaneously under normal MLC conditions, it had no effect when added to uninhibited MLC cells. Conceivably due to neutralization, SEPIR was also incapable of restoring MLC responses conducted in the continuous presence of Class I mAb. Inhibition of MLC responses and reconstitution with SEPIR depended on the effectiveness of Class I antigen inhibition.

Factors influencing Rickettsia tsutsugamushi infection of cultured cells.

Several factors which influence Rickettsia tsutsugamushi infectivity of, and replication in, cultured cells have been investigated in order to establish standard laboratory conditions for reliable quantitation and maximization of rickettsial yield and to begin to explore the nature of the relationship between the rickettsia and its host cell. The extent of scrub typhus rickettsial association with host cells was dependent on the rickettsial and target cell concentrations and on the medium in which the infection took place. Brain heart infusion broth, a standard rickettsial diluent for infectivity, was markedly inhibitory. Both rickettsial and host cell replication were dependent on a component(s) supplied by serum to the tissue culture medium; rickettsial multiplication was less affected by small temperature variation (optimum 34 to 37 degrees C) than was host cell growth (optimum 37 degrees C). The antibiotic daunomycin was very useful in selectively inhibiting host cell replication and permitting attainment of somewhat greater rickettsial yields than in uninhibited cells. These findings have underscored the close relationship between parasite and infected cell and have led to more predictable, higher yields of rickettsiae in vitro.