Rapid Differentiation Between Nocardia and Streptomyces by Paper Chromatography of Whole-Cell Hydrolysates

Whole-cell hydrolysates were prepared from 58 strains of nocardiae and streptomycetes. Strains morphologically intermediate between the two genera and morphological variants of the same strains were included. Paper chromatograms made from the whole-cell hydrolysates clearly demonstrated meso-diaminopimelic acid as a major constituent of cultures of Nocardia spp., and LL-diaminopimelic acid as a major constituent of cultures of Streptomyces spp. In cultures of ten strains of N. madurae and of three of N. pelletieri, meso-diaminopimelic acid predominated, thereby supporting the assignment of these species to the genus Nocardia.     

Genetic analysis of streptomycin-resistance and-dependence inChlamydomonas

Summary Three non-chromosomal and two chromosomal genes which influence resistance to streptomycin are described. Each of the non-chromosomal factors,sr-500,sr-1500, andsd, exhibits uniparental inheritance, with all progeny receiving the factor when it is carried by the parent of mating-typeplus, and none when it is carried by the mating-typeminus parent. The streptomycin-dependence factor,sd, shows zygotic dominance when contributed by the mating-typeplus parent, but not when coming from the mating-typeminus parent, indicating that the uniparental transmission results from events occurring within the zygote early in maturation and well before meiosis. The chromosomal geneA interacts both with chromosomal and non-chromosomal genes at the biochemical level, but does not alter their patterns of inheritance.With 1 Figure in the TextThis paper is dedicated to ProfessorL. C. Dunn in gratitude to him as teacher and advisor, on the occasion of his retirement.This work was supported by grants from the U.S. Public Health Service and the National Science Foundation. The generosity and interest of ProfessorFrancis J. Ryan in providing laboratory space is gratefully acknowledged, as is the technical assistance of MissFran Yablonsky.     

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

Summary Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.     

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5.5-dimethyl-I-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However. in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-0; which rapidly decomposes forming DMPO-OH.     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)     

Two-bond C-C spin-coupling constants in carbohydrates: effect of structure on coupling magnitude and sign

An empirical projection method is described to predict the magnitudes and signs of two-bond ¹³C-¹³C spin-coupling constants (²J_CC) in aldopyranosyl rings. The method has been applied primarily to the interpretation of ²J_CCC values, although the behavior of ²J_COC has also been examined in light of the new approach, producing results which may prove useful in the conformational analysis of O-glycosidic linkages in oligosaccharides. High-level ab initio calculations of ²J_CC values in model compounds were found to be in agreement with the predictions.