Cytokines involved in monocyte mediated tumor cell death and growth inhibition in serum-free medium.

In a serum-free culture system, the release of TNF, lI-1, lI-6, IFN-alpha, and IFN-beta during interaction of elutriated human monocytes (MO) with human tumor cells (TC) was studied by ELISA-technique. Contributions of these cytokines to inhibition of TC-growth and to induction of TC-death by supernatants (SU) gained from such MO/TC-interaction cultures were investigated using affinity chromatography for removal of individual cytokines. Although the TC used are relatively insensitive to recombinant human TNF, withdrawal of TNF causes 50% to 75% reduction of SU-induced TC-death rates, suggesting that susceptibility to TNF is raised during MO/TC-interaction by the other cytokines. Individual removal of other cytokines does not cause reduction of SU-mediated TC-death. However, combined withdrawal of lI-1 and IFN-alpha/beta causes in 2 of 4 TC-lines significant reduction of TC-death. Combined removal of TNF, IFN-alpha/beta, lI-1, and lI-6 leads to complete prevention of SU-mediated growth inhibitory and lytic effects, suggesting that besides these cytokines other signals are not involved significantly. SU-effects can be mimicked by appropriate combinations of authentic cytokines. The response of TC to SU- or cytokine-exposure is strikingly dependent on TC-density, leading at subconfluent TC-density exclusively to inhibition of growth and at postconfluent TC-density to induction of cell death. The principal effect of SU or cytokine combinations in this context seems to be the activation of growth inhibitory signal transduction pathways leading to TC-death in postconfluent TC-populations exclusively if growth stimulatory pathways are activated at the same time. Mouse L cells do not follow this reaction pattern: Their death is exclusively dependent on the presence of TNF in SU and they die upon SU-exposure at postconfluent as well as at subconfluent cell density.     

Rat platelet phospholipase A2. Kinetic characterization using the monomolecular film technique.

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.     

Antiviral nucleoside diphosphate diglycerides: improved synthesis and facilitated purification.

Cytidine diphosphate diglyceride and its analogs have previously been synthesized by condensing phosphatidic acid with the monophosphomorpholidates of the various nucleosides. Yields have been low and purification of the product has been difficult. We report here an improved method for the synthesis of nucleoside diphosphate diglycerides with potential antiviral activity. Phosphatidic acid was activated with morpholine in the presence of dicyclohexylcarbodiimide to phosphatidic acid morpholidate. This compound was condensed with the 5'-monophosphate of the anti-HIV agents 3'-azido-3'-deoxythymidine, 3'-deoxythymidine or 2',3'-dideoxycytidine, and the monophosphate of the anti-HSV agent acyclovir. The resulting nucleoside diphosphate diglycerides are potential candidates for improved antiviral action when compared to the parent nucleoside analogs. Compared to the older method for the preparation of cytidine diphosphate diglyceride and analogs thereof, the new method has several advantages: reaction times are reduced from several days to several hours and the yield of the reactions is generally increased from 20-40% to between 50 and 80%. In addition, the purification of the compounds is greatly facilitated due to the small amount of phosphatidic acid remaining in the reaction mixture.     

Toxin production by Fusarium species from sugar beets and natural occurrence of zearalenone in beets and beet fibers.

Fifty-five Fusarium isolates belonging to nine species were collected from fungus-invaded tissue of stored sugar beets and identified as F. acuminatum (11 isolates), F. avenaceum (1 isolate), F. culmorum (1 isolate), F. equiseti (23 isolates), F. graminearum (4 isolates), F. oxysporum (1 isolate), F. solani (4 isolates), F. sporotrichioides (7 isolates), and F. subglutinans (2 isolates). All isolates were cultured on autoclaved rice grains and assayed for toxicity by feeding weanling female rats the ground-rice cultures of the isolates in a 50% mixture with a regular diet for 5 days. Fifty-eight percent of the isolates were acutely toxic to rats, 26% caused hematuria, 18% caused hemorrhages, and 29% caused uterine enlargement. In most cases, toxicity could not be accounted for by the known toxins found. The following mycotoxins were found in extracts of the rice cultures: zearalenone (22 to 6,282 micrograms/g), chlamydosporol (HM-8) (68 to 4,708 micrograms/g), moniliformin (45 to 400 micrograms/g), deoxynivalenol (10 to 34 micrograms/g), 15-acetyldeoxynivalenol (5 to 10 micrograms/g), diacetoxyscirpenol (22 to 63 micrograms/g), monoacetoxyscirpenol (21 to 26 micrograms/g), scirpenetriol (24 micrograms/g), T-2 toxin (4 to 425 micrograms/g), HT-2 toxin (2 to 284 micrograms/g), neosolaniol (2 to 250 micrograms/g), and T-2 tetraol (4 to 12 micrograms/g). F. equiseti was the predominant species found on visibly molded beets in the field. Six of 25 moldy sugar beet root samples collected in the field contained zearalenone in concentrations ranging between 12 and 391 ng/g, whereas 10 samples from commercial stockpiles were negative for zearalenone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

Lipid conjugates of antiretroviral agents: release of antiretroviral nucleoside monophosphates by a nucleoside diphosphate diglyceride hydrolase activity from rat liver mitochondria.

The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3'-deoxythymidine, 3'-deoxythymidine and 2',3'-dideoxycytidine from the corresponding nucleoside diphosphate diglycerides as a result of rat liver mitochondrial enzymatic activity is shown. The three analogs appeared to be about equally active as substrate for this pyrophosphatase activity which showed maximum conversion rates of 3-6 nmol min-1 mg protein-1 at substrate concentrations between 500 to 800 microM. These results may contribute to the biochemical explanation for the observed anti-HIV activity of this type of phospholipid conjugates in vitro.     

Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells.

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.     

Thermal stability of turnip yellow mosaic virus RNA: effect of pH and multivalent cations on RNA deaggregation and degradation.

Light scattering studies of RNA isolated from turnip yellow mosaic virus (TYMV) revealed a molar mass of 1.9.10(6) g mol-1, which is close to the value of 2.0.10(6) g mol-1 published for intact genomic TYMV RNA (2M RNA). However, gel electrophoresis under denaturing conditions demonstrated that only 30-40% of this native RNA was 2M RNA. Sucrose gradient centrifugation revealed the occurrence of a series of smaller RNA size classes, the mass ratios of which were greatly influenced by the pH of the solution and the presence of EDTA. These results suggest that native TYMV RNA preparations originally contain a mixture of intact RNA particles and of aggregates of RNA fragments with the same molar mass of about 2.10(6) g mol-1, and that the size classes are intermediates in the deaggregation process of the degraded genomic TYMV RNA. The native RNA displayed pH-dependent deaggregation and degradation. The degradation process of 2M RNA followed (pseudo) first-order kinetics. Lower degradation rates were observed for RNA depleted of divalent cations and polyamines. For depleted 2M RNA an enthalpy of activation of about 100 kJ mol-1 and an almost zero entropy of activation was calculated. Similar values were also found for depleted E. coli ribosomal RNAs and depleted MS2 RNA, demonstrating that all RNAs are equally vulnerable to degradation. In the presence of multivalent cations the activation enthalpy for 2M TYMV RNA degradation increased to 150 kJ mol-1 and the entropy of activation to 150 J K-1 mol-1, indicative for a different degradation mechanism.