The interaction of ethanol and exogenous arachidonic acid in the generation of extracellular messengers by mouse peritoneal macrophages

It is increasingly recognized that macrophages play a crucial role in the development of chronic inflammatory states such as alcoholic liver disease. These cells can metabolize free arachidonic acid in the absence of a discernible trigger. The present study was undertaken to examine the short-term effects of ethanol on the generation of these exogenous arachidonate-derived extracellular mediators. Ethanol caused a dose-dependent decrease in the production of both cyclooxygenase and lipoxygenase metabolites. Similar effects were observed on the esterification of exogenous arachidonate into cellular lipids. To characterize further the effects of ethanol on exogenous arachidonic acid metabolism, we studied the short-term responses displayed by macrophages challenged with another soluble stimulus; the tumor-promoting agent phorbol myristate acetate. We observed an inhibition by ethanol of the superoxide anion response triggered by phorbol myristate acetate similar to that observed for exogenous arachidonate oxygenation. Our results show that ethanol can inhibit these soluble stimuli-elicited responses, possibly through its disorganizing effect on plasma membrane.     

Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation

Between 1982 and 1986 51 patients were treated with ciclosporin a (CSA) to prevent graft versus host disease (GvHD) after bone marrow transplantation (BMT). Major side effects of the drug were tremor, hypertension, hepatotoxicity and nephrotoxicity. Acute GvHD 0 degree to II degree occurred in 80% of our patients, and GvHD III degree and IV degree in 20% despite the use of CSA. Two to four days before the onset of GvHD, CSA serum levels were significantly lower on the average in patients who developed GvHD III degree and IV degree compared to the others. Our data indicate that plasma CSA concentrations higher than 250 ng/ml should be achieved to reduce the severity of GvHD after BMT.     

Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

Independence of in vitro iron absorption from mucosal transferrin content in rat jejunal and ileal segments

Isolated non blood-perfused intestinal segments from normal and iron-deficient rats were used in vitro. A modification of the luminal perfusion method according to Fisher and Parsons allowed the comparison of iron and transferrin quantities in the serosal fluid at 15 min intervals. Iron transfer in jejunal and ileal segments was directly proportional to the luminal iron concentration within a dose range of 1 to 100 mumol/l, did not show saturation characteristics and was linear over time. Jejunal segments from iron-deficient rats transferred about twice as much iron as the jejunal controls. In ileal segments there was no difference in iron transfer between iron-deficient and control rats; in both cases transfer amounted to approx. 10% of jejunal controls. An exponential correlation was found, when the decreasing transferrin content of the tissue was plotted against the cumulative water transport. Transferrin and albumin release from jejunal and ileal segments into the absorbate cumulated asymptotically, which is typical for wash-out phenomena. As iron transfer cumulated linearly while transferrin release cumulated in an asymptotic manner, the capacity of transferrin to bind iron ions is exceeded roughly 100 times by molar equivalents of iron in the last absorbate fractions. Independence of iron transfer from mucosal transferrin quantities is concluded. As the molar transferrin/albumin ratios do not show significant differences between plasma and the sequence of absorbate samples, a wash-out from the gut's interstitial space is assumed, which makes plasma the most likely origin of transferrin in the mucosa.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

Purification and characterization of N,N-dimethylformamidase from Pseudomonas DMF 3/3

The N,N-dimethylformamide-hydrolyzing enzyme (DMFase) from Pseudomonas DMF 3/3 has been purified to apparent electrophoretic homogeneity with an overall 49-fold purification, a 24% yield and a final specific activity of 1.98 mumol N,N-dimethylformamide (DMF) hydrolyzed min-1 (mg protein)-1. The native DMFase has a relative molecular mass of 250 000 and is composed of two light-chain (Mr = 15 000) and two heavy-chain (Mr = 105 000) subunits. The stability of DMFase is optimal at pH values above 7.5 and at temperatures below 20 degrees C. The activity of the enzyme is inhibited by metal-chelating agents such as EDTA and 2,2'-dipyridyl. Emission and atomic absorption spectroscopy measurements showed that iron is present in significant amounts in DMFase, indicating that it is an iron-containing amidohydrolase. In the ultraviolet/visible spectrum prominent bands were observed at 224 nm, 280 nm and 396 nm and shoulders are present at 418 nm and 467 nm. DMFase from Ps. DMF 3/3 has an isoelectric point of 7.7. The enzyme exhibits optimal activity between pH 5 and 6 and at 40 degrees C. The substrate spectrum is rather narrow. The enzyme hydrolyzes preferentially substituted short-chain aliphatic amides such as DMF, N-ethylformamide and N-methylformamide. N,N-dimethylformamide, N,N-dimethylacetamide and unsubstituted amides, e.g. formamide, prolinamide, acetamide, acrylamide and butyramide are substrates as well, but are hydrolysed at significantly lower rates. DMFase obeys Michaelis-Menten kinetics and its Km and Vmax values for DMF are 13.8 mM and 1.89 U/mg, respectively, as determined from a Lineweaver-Burk plot.     

Transferrin in isolated cells from rat duodenum and jejunum

Mucosal transferrin was determined as transferrin-like immunoreactivity (TLIR) by means of a 2-site immunoradiometric assay (IRMA). Scraped-off mucosal tissue as well as isolated mucosal cells from the duodenum and jejunum of normal and iron-deficient rats before and after a washing procedure were examined. In iron-deficient rats there was about twice as much TLIR in scraped-off mucosal tissue as in the untreated animals. In the duodenum and jejunum of normal and iron-deficient rats, TLIR contents of the isolated cells in the magnitude of 320-510 ng/mg dry weight were found. Washing isolated cells three times in ice-cold Hank's solution resulted in a nearly tenfold decrease of TLIR content in all groups. In contrast the cells' RNA content remained unchanged.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Investigation by pyrolysis mass spectrometry,phage pattern and plasmid analysis of staphylococci that have reverted from ‘L’-phase to bacterial phase

No major differences have been found in series of Staphylococcus aureus strains which reverted from L-phase, either by pyrolysis mass spectrometry or by phage-typing or sensitivity testing. In L-phase they have been subcultured for a long time or transformed/reverted many times into/from L-phase. Plasmids were lost during transformations/reversions, but there was some difference between the tetracycline-connected plasmids on the one hand and the erythromycin-connected ones on the other.This investigation was supported by the Foundation for Fundamental Research on Matter (F.O.M.), subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).