Quantitative Development and Molecular Forms of Acetyl-and Butyrylcholinesterase During Morphogenesis and Synaptogenesis of Chick Brain and Retina

The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2-3 days; and the development in rhombencephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3-E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14-E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X-100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.6S (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells

Summary We report here that similar to E6-chicken retinal cells, dissociated cells from 5.5-day-old (E5.5) quail retinae reaggregate in rotary culture, multiply about tenfold and reestablish histotypical areas. These cellular aggregates include all nuclear layers either with inversed or correct laminar polarity, depending on the local origin of the cells (called rosetted and laminar in-vitro-retinae (IVR), respectively; Layer and Willbold 1989). In combined cultures, chick and quail cells are evenly mixed only during the first two days of culture. Along with the assembly of single cells into rosettes and then into discrete laminae, sectors of chick and quail cells begin to segregate. They are delineated by borders running radially through all three nuclear layers. Thus, interspecies migration of cells at this advanced stage of differentiation is strongly inhibited. Concomitant with this segregation, coherent radial columns spanning all three layers but containing cells from either species only, can be traced histologically. We conclude that a weak segregation of chick and quail retinal cells takes place already at the single cell level, but that the permanent segregation of entire tissue parts must be due to clonal cellular proliferation within the IVR in conjunction with some developmental-structural mechanism retaining clonal progenies within a columnar order.Abbreviations ECM extracellular matrix - E5.5 days of embryonic age - GCL ganglion cell layer - GC's ganglion cells - i.c. in culture - INL inner nuclear layer - rosetted in-vitro-retina retinal cell organoid aggregated from single cells of the central retina - IPL inner plexiform layer - MRE marginal retinal epithelium - ONL outer nuclear layer - OPL outer plexiform layer - OS ora serrate - PR photoreceptor cell - laminated in-vitro-retina fully laminated retinal cellorganoid resembling an E15-retina aggregated from cells of the eye periphery including RPE - RPE retinal pigment epithelium     

Profiles of eicosanoid production from 14C-arachidonic acid and prostaglandin metabolism by rabbit esophageal mucosa and muscularis

In an attempt to elucidate the possible involvements of eicosanoids in esophageal functions and disorders, we have investigated the formation of both cyclooxygenase and lipoxygenase metabolites from 14C-arachidonic acid by rabbit esophageal tissues. Homogenates of rabbit esophageal mucosa and muscularis were incubated with 14C-arachidonic acid and after ether extraction eicosanoids were separated and quantified by reverse phase high performance liquid chromatography. The predominant cyclooxygenase products were 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 for mucosa and 6-keto-PGF1 alpha, and PGE2 for muscularis. The formation of these products was inhibited both by indomethacin and the dual pathway inhibitor, nordihydrogualaretic acid (NDGA). In mucosa the major eicosanoid was 12-HETE (12-hydroxyeicosatetraenoic acid) which was inhibited by NDGA but not by indomethacin which on the contrary enhanced its formation. Additionally four polar products were synthesized which appeared to be lipoxygenase-dependent as their formation was inhibited by NDGA but not by indomethacin. Muscularis produced as a minor lipoxygenase product only 12-HETE, which was inhibited by NDGA but unchanged in the presence of indomethacin. In addition, both tissues, but mucosa more than muscularis, possessed large prostaglandin catabolizing capacity. The present findings indicate that rabbit esophageal tissues can convert 14C-arachidonic acid into lipoxygenase as well cyclo-oxygenase products which may have a role in esophageal physiology and pathophysiology.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

A drug used in traditional medicine, harpagophytum procumbens: no evidence for NSAID-like effect on whole blood eicosanoid production in human.

Devil's Claw (Harpagophytum procumbens), an herbal product being marketed in Canada and in Europe as a home remedy for the relief of arthritic disease, was investigated in healthy humans on eicosanoid production during spontaneously blood clotting. Volunteers took H. procumbens (daily 4 capsules of 500 mg powder containing 3% of total glucoiridoids) for a period of 21 days. The following are the results (mean (SEM)): before H. procumbens intake, prostaglandin (PG)E2 (ng/ml serum): 2.1 (0.4) (n = 25), thromboxane (TX)B2: 147 (27) (n = 25), 6-keto-PGF1 alpha: 4.4 (0.7) (n = 13), leukotriene (LT)B4: 3.4 (0.4) (n = 25); after intake: PGE2: 3.2 (0.6), TXB2: 143 (24), 6-keto-PGF1 alpha: 4.2 (0.9), LTB4: 3.8 (0.6). Each subject serving as her own control, no statistically significant differences were observed between before and after H. procumbens intake. These results indicate that Devil's Claw lacks, at least in healthy humans and under the selected conditions, the biochemical effects on arachidonic acid metabolism of antiarthritic drugs of the non-steroidal antiinflammatory type.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Evaluation of data transformations used with the square root and schoolfield models for predicting bacterial growth rate.

A comparison was made between mathematical variations of the square root and Schoolfield models for predicting growth rate as a function of temperature. The statistical consequences of square root and natural logarithm transformations of growth rate use in several variations of the Schoolfield and square root models were examined. Growth rate variances of Yersinia enterocolitica in brain heart infusion broth increased as a function of temperature. The ability of the two data transformations to correct for the heterogeneity of variance was evaluated. A natural logarithm transformation of growth rate was more effective than a square root transformation at correcting for the heterogeneity of variance. The square root model was more accurate than the Schoolfield model when both models used natural logarithm transformation.     

Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.