Absence Of Supernatant Research Articles

P105 In vitro interaction of Malassezia and commensal Staphylococcus species

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objective Malassezia is the most abundant fungal skin commensal organism, representing 50%–80% of total fungi present on the skin. It has been associated with many skin disorders such as pityriasis versicolor (PV) and seborrheic dermatitis/dandruff (SD/D). The role of Malassezia in disease manifestation is not discerned. It is important to understand its interaction with bacterial flora such as Staphylococcus epidermidis and S. capitis in vitro. We have studied the interaction of Malassezia and Staphylococcus species isolated from skin flora.Methods Malassezia restricta, M. globosa (n = 5) isolated from patients with SD and M. furfur (n = 5) isolated from PV were sub-cultured on Modified Dixon's agar (MDA). Staphylococcus epidermidis and S. capitis were isolated from patients with SD and sub-cultured on brain heart infusion (BHI) agar. Malassezia species requires media supplemented with lipids (MDA) for its growth. Bacteria and Malassezia were quantified on MDA and BHI agar by Miles and Mishra method to perform interaction between them. For direct interaction, suspensions (100 μl) of M. restricta, M. globose, and M. furfur were prepared in normal saline and added to wells on the plates of lawn cultures containing S. epidermidis and S. capitis (107 CFU/ml). Plates were incubated for 12 h at 35°C and observed for zone of inhibition. To investigate the release of antibacterial compounds into the extracellular environment, M. furfur was inoculated in modified Dixon's broth (MDB) and incubated at 35°C for 5 days. Supernatant was collected at 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h of incubation and evaluated for antibacterial activity by agar-well diffusion assay. Effect of cell-free supernatant of Malassezia on growth of bacteria was also monitored by growth kinetics of S. epidermidis for 24 h in the absence and presence of M. furfur supernatant using Epoch-2 microplate spectrophotometer.ResultsMDA supported the growth of bacteria at different cell densities (107-103 CFU/ml count) and incubation time of S. epidermidis and S. capitis was similar on MDA and BHI. Zone of inhibition (ZOI) was witnessed with M. restricta (20.6 ± 3 mm, 21 ± 3 mm), M. globosa (21 ±1 mm, 22.6 ±2 mm) and M. furfur isolates (16.5 ± 1 mm, 18 ± 2 mm) against S. capitis and S. epidermidis respectively by direct interaction. Inhibition of bacteria by M. furfur was noted from 48-120 h as ZOI (21.7 ±5.1 mm) was observed on bacterial lawn cultured plate. When growth kinetics of S. epidermidis was monitored in presence of M. furfur supernatant, maximum value reached upto 0.26 ± 0.019 only from 0.01 ± 0.001 at OD600 in 9 h including lag phase of 4 h (Fig. 1). However, OD600 value reached upto 0.97 ± 0.005 in 8 h including lag phase of 1.5 h in absence of supernatant. Doubling time calculated from logistic growth equation was 76.6 ± 4.4 and 65.2 ± 2.9 minutes in the presence and absence of supernatant respectively.ConclusionInhibition of bacteria by Malassezia species noted in our study has not been reported earlier. The possible production of antibacterial compounds by Malassezia might be responsible for dysbiosis leading to disease.

Reactivity Differences of Rieke Zinc Arise Primarily from Salts in the Supernatant, Not in the Solids.

Contrary to prevailing thought, the salt content of supernatants is found to dictate reactivity differences of different preparation methods of Rieke zinc toward oxidative addition of organohalides. This conclusion is established through combined single-particle microscopy and ensemble spectroscopy experiments, coupled with careful removal or keeping of the supernatants during Rieke zinc preparations. Fluorescence microscopy experiments with single-Rieke-zinc-particle resolution determined the microscale surface reactivity of the Rieke zinc in the absence of supernatants, thus pinpointing its inherent reactivity independent of the convoluting supernatant composition. In parallel experiments, scanning electron microscopy, energy-dispersive spectroscopy, X-ray photoelectron spectroscopy, and inductively coupled plasma-mass spectrometry characterized the zinc metal chemical composition at the bulk and single-particle levels. Proton nuclear magnetic resonance spectroscopy kinetics characterized bench-scale Rieke zinc reactivity in the presence and absence of different supernatants and exogenous salt additives. Together, these experiments show that the differences in reactivity from sodium-reduced vs lithium-reduced Rieke zinc arise from the residual salts in the supernatant rather than the differing salt compositions of the solids. This supernatant salt also determines the structure of the ultimate organozinc product, generating either the diorganozinc or monoorganozinc halide complex. That different organozinc complexes formed upon direct insertion to different preparations of Rieke zinc was not previously reported, despite Rieke zinc's widespread use. These findings impact organozinc-reagent and nanomaterial synthesis by showing that, unexpectedly, desired Rieke zinc reactivity can be achieved through simple addition of soluble salts to solutions that were used to prepare the metals─a substantially easier synthetic manipulation than solid composition and morphology control.

Impact of Factors Secreted by Tumor Cells on Response of Pleural Mesothelial Cells to Different Sclerosing Agents in an In Vitro Model.

BackgroundChemical pleurodesis is one of the major therapeutic options for patients with recurrent malignant pleural effusion. Mesothelial cells are considered to play a pivotal role in the response to different chemical compounds (sclerosants) used for pleurodesis. Malignant cells might have an impact on the mesothelial response to applied sclerosing agents and, in consequence, on the efficacy of pleurodesis. We aimed to evaluate the impact of cancer cell paracrine on mesothelial cell response to different sclerosing agents.Material/MethodsThe study used mesothelial cell (MeT-5A) cultures stimulated with sclerosing agents (talc, doxycycline, iodopovidone, and TGF-β for 24 h) in the presence or absence of supernatants from adenocarcinoma cultures (HCC827). The mesothelial mRNA expression and protein levels of IL-6, IL-8, and TGF-β was assessed. Further, lung fibroblasts were cultured with and without cell supernatants from previously established cell cultures for 24 h. Then, concentration of soluble collagen was evaluated in culture supernatants.ResultsThe exposure of mesothelial cells to sclerosants decreased the concentration of IL-6 and IL-8 protein. The addition of mediators secreted by adenocarcinoma altered the inflammatory response of the mesothelial cells to sclerosing agents. IL-8 concentration in cultures stimulated with talc and adenocarcinoma supernatant was higher compared to cultures stimulated with talc only. The exposure of lung fibroblasts to supernatant from mesothelial cell (with or without adenocarcinoma) did not affect collagen secretion.ConclusionsAn addition of soluble factors produced by adenocarcinoma altered the inflammatory response of the pleural mesothelial cells after stimulation with sclerosing agents. Our observations suggest that the tumor paracrine effect affects biological pathways of pleurodesis.

Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2\u2013CH3 IgG spacer domains

BackgroundDuring infection with human cytomegalovirus (HCMV) several viral proteins occur on cell surfaces in high quantity. We thus pursue an HLA-independent approach for immunotherapy of HCMV using chimeric antigen receptors (CARs) and bispecific BiTE® antibody constructs. In this context, HCMV-encoded proteins that mediate viral immune evasion and bind human IgG might represent particularly attractive target antigens. Unlike in observations of similar approaches for HIV and hepatitis B and C viruses, however, HCMV-infected cells develop a striking resistance to cytotoxic effector functions at later stages of the replication cycle. In our study we therefore wanted to test two hypotheses: (1) CAR T cells can efficiently inhibit HCMV replication independently from cytotoxic effector functions, and (2) HCMV can be targeted by CH2–CH3 IgG spacer domains that contain mutations previously reported to prevent exhaustion and to rescue CAR T cell function in vivo.MethodsReplication of GFP-encoding recombinant HCMV in fibroblasts in the presence and absence of supernatants from T cell co-cultures plus/minus cytokine neutralizing antibodies was analyzed by flow cytometry. CARs with wild type and mutated CH2–CH3 domains were expressed in human T cells by mRNA electroporation, and the function of the CARs was assessed by quantifying T cell cytokine secretion.ResultsWe confirm and extend previous evidence of antiviral cytokine effects and demonstrate that CAR T cells strongly block HCMV replication in fibroblasts mainly by combined secretion of IFN-γ and TNF. Furthermore, we show that fibroblasts infected with HCMV strains AD169 and Towne starting from day 3 have a high capacity for binding of human IgG1 and also strongly activate T cells expressing a CAR with CH2–CH3 domain. Importantly, we further show that mutations in the CH2–CH3 domain of IgG1 and IgG4, which were previously reported to rescue CAR T cell function by abrogating interaction with endogenous Fc receptors (FcRs), still enable recognition of FcRs encoded by HCMV.ConclusionsOur findings identify HCMV-encoded FcRs as an attractive additional target for HCMV immunotherapy by CARs and possibly bispecific antibodies. The use of specifically mutated IgG domains that bind to HCMV-FcRs without recognizing endogenous FcRs may supersede screening for novel binders directed against individual HCMV-FcRs.

Human trophoblasts cells modulate the occurrence of apoptosis in monocytes infected by Toxoplasma gondii

s / Placenta 36 (2015) 469e521 520 Methods: For this purpose, HTR-8/SVneo cells were treated or not with the supernatant from uninfected or infectedmacrophages and then infected or not by T. gondii. Cytokine secretion and soluble FasL were analyzed by ELISA, apoptosis index and Fas/CD95 expression were determined by flow cytometry and intracellular parasite proliferation was analyzed by enzymatic assay. Results: IL-6 secretion by HTR-8/SVneo cells was synergistically increased with both stimuli, supernatant from macrophages and T. gondii infection. The apoptosis index of infected HTR-8/SVneo cells was decreased in the presence or absence of supernatant from infected macrophages. On the other hand, the apoptosis of infected HTR-8/SVneo cells increased when these cells were treated with supernatant from uninfected macrophages. Also, a low expression of Fas/CD95 and a high soluble FasL release were observed in these conditions. Finally, we did not verify any change in the proliferation of T. gondii. However, there is down-modulation of apoptosis in HTR-8/SVneo cells by the parasite, which probably relates to favoring its establishment in the host cell, while the functional activities of macrophages toward restoring these cell death rates, keep gestational events of invasion and placentation appropriate. Conclusions: All together, these results contribute to better understanding the mechanisms of cellular communication between the extravillous trophoblast cells and macrophages, and demonstrate that infection with T. gondii can interfere with this interaction through the modulation of cell death by apoptosis. PB.47. HUMAN TROPHOBLASTS CELLS MODULATE THE OCCURRENCE OF APOPTOSIS IN MONOCYTES INFECTED BY TOXOPLASMA GONDII A.S. Castro , F.C. Oliveira , P.S.G. Gois , C.M.O.S. Alves , M.B. Angeloni , P.M. Guirelli , R.J. Silva , P.S. Franco , A.O. Gomes , B.F. Barbosa , T.W.P. Mineo , E.A.V. Ferro . 1 Institute of Biomedical Science, Histology and Embryology Department, Federal University Uberlandia, Uberlandia, Brazil; 2 Institute of Biomedical Science, Pharmacology Department, Federal University Uberlandia, Uberlandia, Brazil; 3 Institute of Biomedical Science, Immunoparasitology Department, Federal University Uberlandia, Uberlandia, Brazil Human trophoblast cells modulate the occurrence of apoptosis in monocytes infected by Toxoplasma gondii Objectives: a) to evaluate the role of trophoblast cells (BeWo) in monocyte (THP-1) activity, specifically apoptosis, in the presence or absence of T. gondii infection; b) to investigate whether BeWo cells alter the expression of death receptor (CD95) in THP-1 cells; c) to investigate FasL release by BeWo and THP-1 cells as well as in THP-1 cells stimulated with supernatant from BeWo cells. Methods: THP-1 cells were stimulated with supernatants of BeWo cells previously infected or not with T. gondii, and then infected with parasites. Supernatant of both cells was collected and analyzed for FasL secretion by ELISA. Furthermore, the occurrence of apoptosis in THP-1 cells and the expression of death receptor (CD95) in these cells were evaluated by flow cytometry. Results: The apoptosis index in THP-1 cells stimulated with supernatants of BeWo cells was higher than in untreated THP-1 cells. In the group of infected THP-1 cells, the apoptosis index decreased at all conditions when compared to the group of uninfected THP-1 cells. However, the expression of death receptor (CD95) was higher in the group of infected THP-1 cells and the stimulus with supernatants of BeWo cells induced a higher expression of this receptor in THP-1 cells. Finally, the secretion of FasL was higher in the group of infected THP-1 cells and supernatant of BeWo cells induced the reduction of FasL secretionwhen compared to controls in both groups. Conclusions: Trophoblast cells and T. gondii alter the occurrence of apoptosis, expression of death receptor (CD95) and secretion of FasL in monocytes. These data can be associated with an immune response established by trophoblast cells and monocytes to protect the fetus against infection, although these findings can also represent a potential survival mechanism of the parasite. PB.48. CHLORPYRIFOS EXPOSURE INDUCES TROPHOBLAST BARRIER AND STROMA ALTERATIONS IN HUMAN CHORIONIC VILLOUS EXPLANTS M.E. Ridano , A.C. Racca , J.B. Flores-Martin , E. Bevilacqua , S. GentiRaimondi , R.E. Fretes , G.M. Panzetta-Dutari . CIBICI-CONICET, Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional C ordoba, Argentina; 2 Institute of Biomedical Sciences, University of S~ ao Paulo, S~ ao Paulo, Brazil; Departmento de Histologia y Embriologia, Facultad de Medicina, Universidad Nacional

Impaired CD4+ T cell stimulation of NK cell anti-fibrotic activity may contribute to accelerated liver fibrosis progression in HIV/HCV patients

HIV/HCV co-infection is characterized by a faster progression to liver fibrosis compared to HCV mono-infection. Epidemiologic studies found an association between low CD4(+) T cell counts and advanced stages of liver fibrosis. However, the mechanisms underlying this association remain unclear. CD4(+) T cells critically modulate NK cell activity. Of note, NK cells have been shown to display anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Thus, we speculated that CD4(+) T cells might modulate fibrosis progression by interacting with NK cells. NK cells from HCV(+) (n=35), HIV(+)/HCV(+) (n=28), HIV(+) (n=8) patients, and healthy controls (n=30) were used in this study. NK cells were cultured in the presence or absence of supernatants from CD3/CD28-stimulated CD4(+) cells. Then, NK cells were co-incubated with activated HSC and studied for degranulation, IFN-γ secretion, and induction of HSC apoptosis. Following incubation with CD4(+) T cell supernatants, NK cells displayed a significantly increased activity against primary HSC as compared to unstimulated NK cells. This effect was, at least in part, mediated via an IL-2 dependent upregulation of NKG2D expression. HCV/HIV co-infection was associated with an impaired IL-2 secretion of CD4(+) T cells resulting in an ineffective stimulation of anti-fibrotic NK cell function. Here, we show that CD4(+) T cells are able to stimulate anti-fibrotic NK cell activity via IL-2 mediated upregulation of NKG2D. HIV-induced loss of CD4(+) T cells together with an impaired activity of CD4(+) T cells may contribute to accelerate progression of liver fibrosis observed in co-infection.

SYBR Gold and SYBR Green II are not mutagenic in the Ames test

Favorable photo-physical properties and high affinity to nucleic acids make new fluorescent cyanine dyes of the SYBR-type particularly useful for DNA and RNA visualization. The growing popularity of SYBR-type dyes is also explained by the fact that removal of the dye from the nucleic acids by ethanol precipitation is more efficient and less time-consuming than the phenol–chloroform extraction applied for the widely used phenanthridine DNA stain, ethidium bromide. To evaluate the safety of nucleic acid staining by SYBR Gold and SYBR Green II we compared the mutagenicity of these compounds, with characteristics corresponding to those of ethidium bromide, by use of the Salmonella/mammalian microsome reverse-mutation assays (Ames test). SYBR Green II and SYBR Gold did not show mutagenicity either in frame-shift or in base-substitution indicator strains, TA98 and TA100, respectively. These results were observed both in the presence and in the absence of supernatant from a rat-liver homogenate S9. Mutagenicity of these stains was not observed although their toxic concentration was reached. Toxic effects of SYBR Green II and SYBR Gold were seen approximately at the same molar concentrations as reported previously for SYBR Green I. As expected, ethidium bromide revealed strong mutagenicity with a maximum increase of 60-fold above the vehicle controls in the frame-shift indicator strain TA98 in the presence of rat-liver S9 extract. Thus, SYBR Gold and SYBR Green II do not show mutagenicity in our tests, even at toxic doses, and these DNA stains represent safer alternatives to ethidium bromide for nucleic acid visualization.

Dose and Timing Limitations in the Use of Bone Marrow-Derived Mesenchymal Stem Cells for the Treatment of Experimental Arthritis.

The recently recognized potential of mesenchymal stem cells (MSCs) to differentiate into a broad spectrum of tissues and to act as immune regulators beyond the barriers of embryonic germ layers and major histocombatibility comlex (MHC) restriction, has emerged intense research interest on their possible use in a broad spectrum of clinical entities. Although the immunoregulatory potential of MSCs has been shown to effectively control GvHD in several preclinical and clinical studies, their role in autoimmune diseases has not been extensively explored in animal models. The goal of this study was to investigate the in vitro effect of rat bone marrow-derived MSCs on cultured fibrobIast-like synoviocytes (FLS) and T-cells from the spleen after induction of adjuvant arthritis (AA) by FCA as well as their in vivo effect in a rat model of AA resembling human rheumatoid arthritis. MSCs were isolated from bone marrow and were characterized by CD45 negativity and CD54, CD29 positivity in FCM analysis. Differentiation assays were performed to confirm their adipogenic, osteogenic and chondrogenic potential. Culture of AA-FLS in the presence of supernatant from syngeneic (syng) or allogeneic (allo) MSCs at passage 2–3, reduced the AA-FLS (p<0.022) and the ConA-stimulated AA-T-cell (p=0.04) proliferation in a dose-dependent manner, as compared to AA-FLS or AA-T-cell proliferation in the absence of supernatant. Cell-to-cell contact by coculture of activated T-cells with syng or allo MSCs produced a stronger inhibition over the supernatant (p<0.0001), in all tested MSCs dilutions and even at the lowest MSCs :T-cell ratio of 0.05:1. The inhibitory effect of allo as compared to syng MSCs in activated AA T-cells, was stronger both by secreted agents (p=0.017) or by cell to cell contact (p=0.0001). In vivo, low doses of syng MSCs (0.5-5x10^5cell/recipient) administered iv, intrasplenic or intrabone marrow, at single or multiple infusions, didn't significantly reduce the disease score of MSC-treated as compared to control rats. In contrast, repeated, higher dose (6x10^6cell/recipient), iv infusions of syng or allogeneic MSCs from male donors (Y+MSCs) to female recipients, before the onset of AA (d4 and d9 post AA induction) resulted in significantly lower arthritic scores when compared to control animals. MSC-treated animals preserved a rather normal joint architecture with focal synovial hyperplasia, limited pannus formation and without bone destruction or chondroplasia. In contrast, the joints of arthritic control rats, appeared with a thickened synovial membrane, erosive pannus and dense inflammatory cell infiltration, chondroplasia and osteoplasia. Reduced presence of CD3+, CD11b+, NF-kb+ cells and less intense angiogenesis (FVIII+cells) was demonstrated by immunohistochemistry in the synovium of transplanted rats as compared to the control group. No Y+MSCs were detected in the spleen, bone marrow or in cultured FLS from the synovial membrane at day30 post AA induction, by PCR (sry gene), immunohistochemistry (sry protein) or FISH (Y chromosome), suggesting that the observed benefit was mostly a result of immunomodulation not derived by MSCs homing to target tissues, or migration of MSCs to target tissues may have occured earlier. On the other hand, when the same cell dose was injected after the onset of arthritis (d13 and d20 post AA induction) no clinical benefit could be observed. Our data suggest that MSCs may represent a new therapeutic approach for autoimmune arthritis, however, due to dose and timing limitations in their use, further studies are needed to clinically exploit this potential.

Macrophage Migration Inhibitory Factor Is Up-Regulated in Human First-Trimester Placenta Stimulated by Soluble Antigen of Toxoplasma gondii, Resulting in Increased Monocyte Adhesion on Villous Explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAg, interferon-gamma (IFN-gamma), or STAg plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAg (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAg or STAg plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Phagocytosis of Giardia lamblia trophozoites by human colostral leukocytes

To determine the phagocytic activity of the polymorphonuclear and mononuclear cells present in human colostrum, and to verify the influence of opsonins in the adherence, ingestion and killing of Giardia lamblia trophozoites. Polymorphonuclear and mononuclear phagocytes were incubated with G. lamblia trophozoites, in the presence as well as the absence of supernatant of human colostrum (the source of opsonins) for 30, 60 and 120 min. The trophozoites/phagocytes ratio was 1:1, and the percentage of phagocytosed trophozoites was determined by microscopic examination of acridine orange-stained cells. The mononuclear phagocytes presented more functional activity than the polymorphonuclear. The highest indexes of adherence (77.6 +/- 5.1), ingestion (68.9 +/- 5.5) and killing (48.5 +/- 4.9) were obtained through the incubation of mononuclear cells in the presence of colostrum supernatant for 120 min. The phagocytes of human colostrum were able to ingest G.lamblia trophozoites and presented microbicidal activity in vitro, suggesting that these phagocytes may act as an additional mechanism of protection against infant giardiasis through breastfeeding.

In vitro microtubule-based organelle transport in wild-type Dictyostelium and cells overexpressing a truncated dynein heavy chain.

The transport of vesicular organelles along microtubules has been well documented in a variety of systems, but the molecular mechanisms underlying this process are not well understood. We have developed a method for preparing extracts from Dictyostelium discoideum which supports high levels of bidirectional, microtubule-based vesicle transport in vitro. This organelle transport assay was also adapted to observe specifically the motility of vesicles in the endocytic pathway. Vesicle transport can be reconstituted by recombining a high-speed supernatant with KI-washed organelles, which do not move in the absence of supernatant. Furthermore, a microtubule affinity-purified motor fraction supports robust bidirectional movement of the salt-washed organelles. The plus and minus end-directed transport activities can be separated by exploiting differences in their affinities for microtubules in the presence of 0.3 M KCl. We also used our assay to examine organelle transport in a strain of Dictyostelium overexpressing a 380-kDa C-terminal fragment of the cytoplasmic dynein heavy chain, which displays an altered microtubule pattern (380-kDa cells; [Koonce and Samso, Mol. Biol. Cell 7:935-948, 1996]). We have found that the frequency and velocity of minus end-directed membrane organelle movements were significantly reduced in 380-kDa cells relative to wild-type cells, while the frequency and velocity of plus end-directed movements were equivalent in the two cell types. The 380-kDa C-terminal fragment cosedimented with membrane organelles, although its affinity was significantly lower than that of native dynein. An impaired membrane-microtubule interaction may be responsible for the altered microtubule patterns in the 380-kDa cells.

High concentrations of phosphatidylinositol-4,5-bisphosphate may promote actin filament growth by three potential mechanisms: Inhibiting capping by neutrophil lysates, severing actin filaments and removing capping protein- β2 from barbed ends

Cell locomotion requires rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. Phosphatidylinositol-4,5-bisphosphate (PIP 2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation. We have examined the effects of PIP 2 micelles on the Ca 2+-independent actin filament capping activity in high speed supernatants of neutrophil lysates which we had previously demonstrated to be almost entirely due to capping protein- β 2, a homologue of cap Z. High concentrations of PIP 2 totally prevented the capping of exogenous spectrin-F-actin seeds by dilute supernatants of neutrophil extracts. Capping could also be inhibited, albeit less effectively, by PIP and PI, but not by other phospholipids. When incubated with filaments in the absence of supernatant, PIP 2 increased the number of growing ends. PIP 2 also uncapped previously capped actin filaments, as demonstrated by incubating supernatant-capped and uncapped seeds with and without PIP 2 and then comparing the initial elongation rates after addition of pyrenyl-G-actin. Incubation of capped seeds with high concentrations of PIP 2 increased the number of free barbed ends to a level comparable to that of the uncapped seeds exposed to PIP 2. PIP 2 caused uncapping to occur too quickly to be explained simply by the off-rate of capping protein- β 2, implying that PIP 2 interacted directly with capping protein on the filament ends. In fact, PIP 2 transiently uncapped capped seeds in the presence of excess free capping protein. From our data, we estimate that millimolar concentrations of PIP 2 (almost 100-fold higher than the amount predicted from the effective concentration in purified systems) would be required to inhibit all the capping protein- β 2 in the cytosol. This discrepancy probably results, in large part, from sequestration of PIP 2 by other PIP 2-binding proteins in the cytoplasm. If PIP 2 mediates differential cytoskeletal growth after chemoattractant stimulation in vivo, very high concentrations may be required subjacent to the plasma membrane for regional severing and uncapping of actin filaments to occur quickly near the perturbed membrane.

Full Scale Optimization of Biological Phosphorus Removal at Kelowna, Canada

A newly constructed biological nutrient removal treatment plant operating in the Phoredox mode was used to test the effects on plant efficiency of varying two important operating conditions - the point of introduction of return sludge flow, and the addition of primary sludge thickener supernatant to the bioreactor. Overall treatment results for Kelowna's weak sewage showed that the effluent was consistently below 10 mg/L BOD and 4 mg/L suspended solids, while total P was below 2 mg/L 94 percent of the time, and total N was below 6 mg/L 90 percent of the time. There was no significant difference in effluent nutrient quality between tests with all return sludge entering the anaerobic zone or with half entering the anaerobic zone and half entering the first anoxic zone. Primary sludge thickener supernatant added to the anaerobic zone typically resulted in an effluent P concentration of less than 0.1 mg ortho-P/L, while the absence of supernatant produced an effluent containing greater than 2 mg ortho-P/L.

Isolation of species-specific and stage-specific adhesion promoting component by disaggregation of intact sea urchin embryo cells

The supernatant obtained from dissociation of Strongylocentrotus purpuratus blastula embryos effectively promoted reaggregation of S. purpuratus blastula cells by more than 3-fold over that in the absence of supernatant. The aggregation enhancing effect was completely species-specific and developmental stage-specific. The supernatant enhanced aggregation of glutaraldehyde-fixed cells, suggesting that it acts at the cell surface. Trypsinization of blastula cells did not affect the ability of dissociation supernatant to promote reaggregation, whereas trypsin treatment of dissociation supernatant completely destroyed reaggregation enhancing activity. We feel that the attributes of this system are several: (1) future biochemical work should be informative because liters of active supernatant containing about 1 mg protein/ ml can easily be obtained; (2) all experiments are done in sea water, the natural medium of the cells resulting in experimental manipulations that are not greatly different from the normal living conditions of the cells; (3) the isolated adhesion-promoting component is species-specific and stage-specific and appears to act at the cell surface.

948 GENERATION OF CONCANAVALIN A (Con-A)-INDUCIBLE SUPPRESSIVE FACTORS IN ACUTE LYMPHOCYTIC LEUKEMIA (ALL)

Leukemic blasts (LB) obtained at diagnosis from 6 non-T, non-B, and 2 T-cell patients with ALL were studied for their ability to suppress or enhance the proliferative capacity of normal allogeneic peripheral blood lymphocytes (PBL). LB or control lymphocytes (Ly), preincubated at 37°C for 48 hrs with 100 μg/ml Con-A (Ca) or without Con-A (Co), were treated with mitomycin-C. One hundred thousand Ca- or Co-treated LB or Ly were added to 1 × 105 PBL and cultured with or without supernatants of preincubated cultures in the presence of phytohemagglutinin, Con-A, and pokeweed mitogen. The response of PBL in the presence of supernatants from LB or Ly was markedly suppressed for all stimulations (P = <.001). However, in the absence of supernatants, 2 patients with non-T, non-B ALL and 1 T-cell ALL suppressed the PBL response for all mitogens (% suppression range 10-73.9; control range 19.3-81.1). One T cell and 4 non-T, non-B ALL showed enhancement (range 11.6-232.0). We conclude that T-cell factors are bifunctional molecules recognizing the stimulant by one end, selecting the cell type by the other end, leading to suppression or augmentation with their restriction specificity.

Desaturation of Oleyl Phospholipid to Linoleyl Phospholipid in Torulopsis utilis

Abstract An enzyme system from Torulopsis utilis consisting of microsomal pellet and supernatant (100,000 x g) catalyzes the conversion of either oleyl-CoA or oleyl phospholipid to linoleate. The desaturation of oleyl phospholipid has been demonstrated both with biosynthetic and chemically synthesized substrate. Incubation of Torulopsis particles with [3H]-oleyl-CoA in the absence of supernatant affords [3H]-oleyl phospholipid. This material is desaturated to linoleate either in situ after addition of supernatant or, after isolation, by the complete enzyme system. Fractionation of the biosynthetic [3H]oleyl lipid indicates that oleyl phosphatidylcholine is the most active of the various phospholipid fractions for desaturation. Synthetic 1,2-di[14C]oleyl phosphatidylcholine is converted to linoleate with high efficiency. Linoleate formation from either biosynthetic or chemically synthesized oleyl phospholipid requires both the particulate fraction and the supernatant from Torulopsis extracts.

Ribosome-catalysed peptidyl transfer: the polyphenylalanine system

The reaction of puromycin with polyphenylalanine-charged Escherichia coli ribosomes has been characterized. Release of polyphenylalanine from transfer RNA was estimated by a new, rapid method based on differential solubilities of substrate and product in m -cresol. Occurrence of a reaction in the absence of supernatant and GTP was demonstrated with (a) polyphenylalanine-charged 70 s ribosomes which had been washed with salt solution under conditions similar to those used for removing the supernatant factors of protein synthesis, and (b) purified, polyphenylalanine-charged 50 s subunits. Only monovalent and divalent cations and buffer are required for this “supernatant-independent” reaction, and under suitable conditions 70 to 80% of the polyphenylalanine is released from transfer RNA. Evidence from these and other results indicates that the puromycin reaction takes place by the same mechanism as peptide bond formation in protein synthesis, and that peptide bond formation is catalysed by the 50 s ribosomal subunit and does not directly involve supernatant factors or GTP. The reaction has characteristics typical of enzyme reactions, and the catalytic agent has been named “peptidyl transferase”. Kinetics of the supernatant-independent reaction are heterogeneous. A fraction of the charged ribosomes is highly reactive and may represent a preformed enzyme-substrate complex. Effects of puromycin concentration and various other factors on the kinetics are described. The reaction is inhibited by addition of 4 m -urea or 0·5% sodium dodecyl sulphate, by prior heating of charged ribosomes to 70°C, and by pre-treatment with 0·5 to 2% formaldehyde. The principle findings with 70 s ribosomes were confirmed with isolated, poly-phenylalanine-charged 50 s subunits.

Studies on the cyclophorase system. XVIII. The oxido-reductions of glycolysis

Summary The oxidative enzymes of glycolysis are present in cyclophorase preparations. Their activity is not increased by the presence of pyridine nucleotides. When a small quantity of supernatant is added to the gel, presumably to supply the nonoxidative enzymes, it can oxidize glucose and various intermediates of the glycolysis scheme to carbon dioxide and water. D (-)-Lactic acid is oxidized by cyclophorase preparations in the absence of supernatant, but supernatant is required for the oxidation of L (+)-lactic acid.