The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells. NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner.

Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.     

Vine Deloria Jr. as a Philosopher of Education: An Essay of Remembrance

This essay engages the concepts of maturity, relationality, and responsibility in the writings of Vine Deloria Jr. as foundational to a Native philosophy of education. After situating Deloria and these Native philosophic concepts as a moment of difference in the colonial—modern world, I explore how these concepts of maturity, relationships, and responsibility have been discussed in his work and remain potent forces in the continuing evolution of education among Native peoples.     

The development of a population of spinal cord neurons and their axonal projections revealed by GABA immunocytochemistry in frog embryos

The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.     

Preparation of scirpentriol and triacetoxyscirpenol in good yield from cultures of Fusarium sambucinum NRRL 13495

Crude extracts of filtrates of cultures of Fusarium sambucinum NRRL 13495 were acetylated or hydrolyzed. After chromatography on cartridge columns of silica gel and recrystallization three times from mixtures of ethyl acetate and hexane, 3,4,15-triacetoxyscirpenol (435 +/- 10 mg/liter of filtrate; mean +/- standard error [n = 3]) and the parent alcohol scirpentriol were isolated (261 +/- 29 mg/liter of filtrate; mean +/- standard error [n = 3]) in 68 and 53% yield for a 130- and 14-fold improvement, respectively, over prior reports.     

Depolarized fluorescence photobleaching recovery

The effects of the fact that the laser sources typically used in fluorescence photobleaching recovery (FPR) experiments in the most commonly employed in-line microscope imaging geometries, are highly linearly polarized, are examined in some detail. The implications of the results, in particular for the interpretation of FPR data in complex cell membrane systems in terms of laterally mobile and immobile sub-populations of the labelled molecular species of concern, are discussed. Methods of experimentally eliminating the potentially major rotational diffusion-based artifacts, different from those appropriate to three-dimensional (solution or suspension) systems which require other than in-line geometries, are delineated.Abbreviations FPR fluorescence photobleaching recovery - FRAP fluorescence recovery after photobleaching - 2- and 3-D two- and three-dimensional     

DNA base changes induced following in vivo exposure of unadapted, adapted or ada- Escherichia coli to N-methyl-N'-nitro-N-nitrosoguanidine

The adaptive response is one of the major repair pathways in Escherichia coli that removes DNA alkylation damage. To investigate the role of the adaptive response in mutagenesis, the E. coli gpt forward mutation assay system was used to determine the mutation spectrum of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in MNNG-adapted and unadapted GP120 (wild-type) and unadapted PJ5 (ada-5) cells. We observed that 34/37 mutations in the unadapted GP120 cells, 38/40 mutations in the adapted GP120 cells, and 10/10 mutations in the PJ5 cells were GC----AT transitions. The remaining 3/37 mutations in the unadapted GP120 cells were large insertions. The remaining 2/40 mutations in the adapted GP120 cells were transversions with one a GC----CG and the other an AT----CG. A surrounding sequence specificity of mutagenesis was observed for the GC----AT transitions in both the unadapted (GP120 and PJ5) and adapted (GP120) cells, with 70% of the unadapted PJ5, 68% of the unadapted GP120, and 61% of the adapted GP120 mutations occurring at the middle G of the sequence 5'--GG(A or T)--3'. Both strains also displayed a statistically significant preference for mutagenesis at guanine bases in the non-transcribed strand. The overall distribution of mutated sites in the gpt gene in adapted and unadapted cells was similar, although the rate of mutations at certain sites appeared different. These minor differences could result from either non-uniform repair of alkylation damage at different sites on the DNA, or altered processing of the alkylated bases to mutations in the adapted state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

In situ detection of early replication phases of a gemini virus in legume protoplasts

Protoplasts of Phaseolus vulgaris L. (Top Crop), infected with bean golden mosaic virus, were isolated and fixed by various methods for in situ hybridization. An iodine-125 labeled probe was made from the replicative form of the virus. The localization and quantitation was done by autoradiography. Cell wall removal lowered the background and allowed a more accurate analysis. RNase was used to eliminate the possibility of hybrids to RNA. The evidence suggests a sequence of virus movements starting from rough endoplasm reticulum, moving to the nuclear membrane, and finally with the highest concentration inside the nucleus.Abbreviations BGMV bean golden mosaic virus - rfBGMV or rfDNA replicative double-stranded DNA virus - ssDNA single-stranded virus     

Enhanced Survival of the Cyanobacterium Oscillatoria terebriformis in Darkness under Anaerobic Conditions

Oscillatoria terebriformis, a thermophilic cyanobacterium, maintained viability in darkness under anaerobic conditions by fermenting exogenous glucose or fructose to lactic acid. The time period of survival was greatly extended when the environmental redox potential was lowered by the addition of sodium thioglycolate or titanium(III) citrate. When exposed to aerobic conditions in darkness, many trichomes underwent lysis in 6 h, and death of all cells occurred in 2 to 3 days. The endogenous aerobic respiration rate was high, and the limited dark aerobic survival period appeared to be due to depletion of stored glycogen. Fructose or glucose did not support or increase aerobic respiration in darkness or lengthen aerobic survival time. Enhanced survival of O. terebriformis in darkness under anaerobic, reducing conditions correlates well with the natural nighttime position of this species within sulfide-rich microbial mats associated with hot springs of western North America.