Effects of PGE1 or PGE2 and/or acetazolamide on expulsion of Nippostrongylus brasiliensis from rats

PGE1 and PGE2 have been reported to enhance natural expulsion of Nippostrongylus brasiliensis, a nematode parasite, from the intestine of the rat. Mucus production may also be a key element of worm rejection. Our study attempts to determine if 1) PGE1 or PGE2 alter the normal course of infection with N. brasiliensis in rats, 2) a known mucous enhancing drug, acetazolamide, can augment the rate of worm expulsion, and 3) combinations of prostaglandins and acetazolamide affect N. brasiliensis in the rat. Rats were inoculated with approximately 1,000 infective larvae of N. brasiliensis. Animals were administered, intraduodenally, one of the following: 0.2 ml 0.9% NaCl; 0.2 ml 100% ethanol; 250 micrograms PGE1/0.2 ml 100% ethanol; 250 micrograms PGE2/0.2 ml 100% ethanol; 250 micrograms acetazolamide/0.2 ml 100% ethanol; 250 micrograms PGE1 or PGE2 + 250 micrograms acetazolamide/0.2 ml 100% ethanol. These solutions were given in a single bolus on day 6 postinoculation (PI) or twice daily on days 6-9 PI. Following these treatments the number of parasite ova per gram feces per day for days 6-10 PI and numbers of worms present at necropsy on day 10 PI were determined. Treatment with prostaglandins or acetazolamide or both failed to adversely affect egg deposition by adult female worms or the number of worms in the small intestine. These results do not support the involvement of prostaglandins in the expulsion of N. brasiliensis from the host intestine.     

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose beta-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55 degrees C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity.     

A model for glucose metabolism in energy-sufficient bacterial cultures

Summary A model for uncoupled glucose uptake under energy-sufficient conditions is presented. The model is derived from glucose catabolic pathways. The resulting model predicts specific glucose uptake rate as a function of both growth rate and extracellular glucose concentration. This prediction is consistent with reported literature data.     

Effect of bacterial growth-inhibiting ingredients on the Ames mutagenicity of medicinal herbs

A solvent fractionation method was introduced to screen for mutagenicity in 10 medicinal herbs being consumed in Korea. The Ames mutagenicity test result of Scutellariae and Rhei was significantly increased by eliminating growth-inhibiting substances through solvent fractionation of the crude extract. It is suggested that a physicochemical pretreatment should reduce the false-negative results which are caused by the presence of growth-inhibiting substances in complex mixtures.     

Electrical injury mechanisms: electrical breakdown of cell membranes

Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electrical field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.     

Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain

Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.     

Reciprocal effects of epidermal growth factor and transforming growth factor beta on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.     

The final step in the de novo biosynthesis of platelet-activating factor. Properties of a unique CDP-choline:1-alkyl-2-acetyl-sn-glycerol choline-phosphotransferase in microsomes from the renal inner medulla of rats

Final steps in the synthesis of platelet activating factor (PAF) occur via two enzymatic reactions: the acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by a specific acetyltransferase or the transfer of the phosphocholine base group from CDP-choline to 1-alkyl-2-acetyl-sn-glycerol by a dithiothreitol (DTT)-insensitive cholinephosphotransferase. Our studies demonstrate that rat kidney inner medulla microsomes synthesize PAF primarily via the DTT-insensitive cholinephosphotransferase since the specific activity of this enzyme is greater than 100-fold higher than the acetyltransferase. The two cholinephosphotransferases that catalyze the biosynthesis of phosphatidylcholine and PAF have similar Mg2+ or Mn2+ requirements and are inhibited by Ca2+. Also topographic experiments indicated that both activities are located on the cytoplasmic face of microsomal vesicles. PAF synthesis was slightly stimulated by 10 mM DTT, whereas the enzymatic synthesis of phosphatidylcholine was inhibited greater than 95% under the same conditions. The concept of two separate enzymes for PAF and phosphatidylcholine synthesis is further substantiated by the differences in the two microsomal cholinephosphotransferase activities with respect to pH optima, substrate specificities, and their sensitivities to temperature, deoxycholate, or ethanol. Study of the substrate specificities of the DTT-insensitive cholinephosphotransferase showed that the enzyme prefers a lipid substrate with 16:0 or 18:1 sn-1-alkyl chains. Short chain esters at the sn-2 position (acetate or propionate) are utilized by the DTT-insensitive cholinephosphotransferase, but analogs with acetamide or methoxy substituents at the sn-2 position are not substrates. Also, CDP-choline is the preferred water-soluble substrate when compared to CDP-ethanolamine. Utilization of endogenous neutral lipids as a substrate by the DTT-insensitive cholinephosphotransferase demonstrated that sufficient levels of alkylacetylglycerols are normally present in rat kidney microsomes to permit the synthesis of physiological quantities of PAF. These data suggest the renal DTT-insensitive cholinephosphotransferase could be a potentially important enzyme in the regulation of systemic blood pressure.     

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.