The stimulation and binding of CTP: phosphorylcholine cytidylyltransferase by phosphatidylcholine-oleic acid vesicles

The activity of the low molecular weight form of cytidylyltransferase from fetal lung cytosol and adult liver cytosol was stimulated more by phosphatidylcholine-oleic acid (1:1 molar ratio) vesicles than by phosphatidylglycerol vesicles. Phosphatidylcholine alone did not stimulate the activity, while oleic acid alone produced only slight stimulation. Vesicles prepared from phosphatidylinositol, phosphatidylglycerol-cholesterol (2:1) and phosphatidylglycerol-phosphatidylcholine (1:1) all stimulated the activity to the same extent. Phosphatidylcholine-oleic acid vesicles (molar ratio 2:1) produced less stimulation than 1:1 vesicles. Phosphatidylcholine-palmitic acid vesicles (2:1) were about 50% as active as the corresponding phosphatidylcholine-oleic acid vesicles. All vesicles were in the size range of small unilamellar vesicles as judged by Sephacryl S-1000 chromatography. Stimulation also occurred when phosphatidylcholine vesicles and oleic acid were added separately to the assay. The stimulation by phospholipid vesicles was correlated with the ability of the vesicles to bind cytidylyltransferase, determined by sucrose density centrifugation of the enzyme-vesicles mixtures. We conclude that the stimulation of soluble cytidylyltransferase occurs through binding of the enzyme to anionic membrane surfaces. Suitable anionic membranes can be prepared either from anionic phospholipids, or by the addition of anionic lipids (unesterified fatty acids or phosphatidylglycerol) to phosphatidylcholine.     

Selective inhibition of the potato scab pathogen by antagonistic bacteria and substrate influence on antibiotic production

Summary In a long-term field experiment a soybean cover crop and green manure incorporation prevented the build-up of common scab of potato whereas barley, employed in the same manner, increased disease incidence. When soil from these plots was assayed for organisms antagonistic toS. scabies, a bacterium indentified asBacillus subtilis, was found to be predominant. Laboratory tests showed thatS. scabies was more sensitive to the antibiotic produced by this bacterium than most non-pathogenicStreptomyces spp which were tested. The antibiotic was found to be similar to bacitracin and activity was expressed as units bacitracin. Water extracts of greenhouse-grown soybean and barley were compared, at different concentrations, as a substrate for growth and antibiotic production by this bacterium. It was found that on the soybean extract, antibiotic activity, as measured by the standard filter-paper-disc technique, was 2.5 to 3 times greater per unit of bacterial growth than when barley extract was used as the substrate. Similar results were obtained when extracts of partially decomposed tissue were used. It is suggested that when evaluating antagonistic organisms as a possible factor in the behavior of plant pathogens in soil, the relative sensitivity of the pathogen to the antibiotic activities of the antagonists as well as the substrates available to the antagonists should be considered.     

Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies.

In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC.     

HIV-1 GP120-mediated immune suppression and lymphocyte destruction in the absence of viral infection

The magnitude of immunologic defects observed in HIV-1-infected individuals before the development of overt AIDS is disproportionately high in comparison to the levels of infectious virus in these patients--suggesting that factors other than direct virus-induced cytopathology may be involved. With this in mind, we investigated the immunologic consequences of the interaction between purified HIV-1 gp120 and the CD4 molecules expressed by uncommitted as well as Ag-specific lymphocytes. HIV-1 gp120 exhibited a dose-dependent immunosuppressive effect on: 1) Ag-driven proliferation of cloned CD4+ lymphocytes, 2) OKT3-driven proliferation of cloned CD4+ lymphocytes, and 3) cytolytic activity of CD4+, EBV-specific CTL. Thus, HIV-1 gp120 can, in a manner similar to OKT4A antibodies, suppress T cell activation and the expression of cytolytic activities through its interaction with CD4. Additionally, activated CD4+ lymphoblasts can be rendered susceptible to immune cytolysis by virtue of their binding of purified gp120. This "targeting" of activated lymphoblasts can occur with levels of gp120 far below that which is needed to saturate all OKT4A-defined CD4 epitopes. Adsorbed gp120 could be demonstrated on the surface of these cells for up to 12 h, a sufficient time for interaction with host cytolytic elements. The data from these in vitro modeling experiments highlight one of many potential mechanisms of HIV-1 induced immunosuppression and lymphocyte destruction that can occur in the absence of infectious virus and that is based on the unique interaction between HIV-1 gp120 and its cellular receptor, CD4.     

Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase.

We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).     

The effect of AZT on in vitro lymphokine-activated killer (LAK) activity in human immunodeficiency virus type-1 (HIV-1) infected individuals

Human immunodeficiency virus type-1 (HIV-1)-infected individuals exhibit functional impairment in various forms of cell-mediated cytotoxicities (CMC) at all stages of disease. The purpose of this study was to determine (i) if peripheral blood mononuclear cells (PBMC) obtained from HIV-1-infected patients could be stimulated in vitro to yield lymphokine-activated killer (LAK) activity; (ii) if non-MHC-restricted gp120-specific CMC could be preserved; and (iii) what effect zidovudine (AZT) would have on LAK activity. Fourteen asymptomatic HIV-1 seropositive adults and five healthy seronegative adults (controls) were evaluated. PBMCs were isolated and incubated in media or supplemented with IL-2 for 4 or 72 hr. Lysis of the NK resistant target cell line, Daudi, was similar for the control and experimental group. The increase in activity after stimulation was elevated to a similar degree in both seronegative and seropositive groups (P less than 0.001). LAK activity was significantly decreased (P = 0.011) when AZT was added to LAK cultures. In addition, virus production may not have been completely inhibited by AZT in LAK cultures. Thus, PBMCs from asymptomatic HIV-1-infected patients could be stimulated to yield LAK activity. However, AZT can impair LAK generation. It is unclear if LAK activation results in virus production that cannot be inhibited by AZT in this system. Further definition in other patient populations is required prior to applying this information to clinical trials.     

Anti-HIV-1 ADCC: clinical and therapeutic implications.

The predominant antienvelope cell-mediated cytotoxicity in HIV-1-infected patients is a direct form of antibody-dependent cellular cytotoxicity (ADCC) in which circulating NK/K cells armed with cytophilic antibodies comprise a cytolytic effector cell complex capable of destroying HIV-1-expressing targets. This non-MHC-restricted form of virus-specific cytotoxicity is present in most infected patients, with maximum activity in early disease, gradually declining with disease progression. This endogenous cytotoxicity provides a focal point in the design of interventive strategies involving immune-based therapies. In the first such attempts, the lymphokine interleukin-2 has been employed in an effort to augment these potentially beneficial cytolytic reactivities. The focus of this article is to present the rationale, early clinical results, and future direction of such therapeutic approaches and, in doing so, to illustrate how careful basic research findings can be applied to the design of rational therapeutic strategies.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.