Lack of evidence for activation of a serum factor in protease-induced differentiation of mouse erythroleukemia cells

summary The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induced up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B⁺). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B⁺ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B⁺ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B⁺ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B⁺ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells. This study was supported by U.S. Public Health Service Grants CA 24403 and CA 37874; American Cancer Society Grant CH-303; the Spingold Foundation; the Chemotherapy Foundation, Inc; the Gar Reichman Foundation; an Irving Alpert Cancer Research Award; and institutional general research funds. Part of this work was presented at the 1984 meeting of the American Society of Biological Chemists and American Association of Immunologists, St. Louis, MO (21).     

Local regeneration in the retina of the goldfish

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transsclearal surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2′-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithiallike cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.     

Physicochemical characterization of carrageenan fromGigartina teedii (Rooth) Lamouroux (Gigartinales,Rhodophyta)

The phycocolloids of female gametophytes ofGigartina teedii (Roth) Lamouroux harvested in Roscoff (Brittany, France) are a hybrid carrageenan resulting from juxtaposition of fragments of kappa-, iota-and nu-carrageenan. They represent 70% of the dry matter of the alga in summer. After alkaline transformation the proportion of iota-carrageenan increased to 76%, demonstrating the presence of nu-carrageenan. Absence of mu-carrageenan, the precursor of kappa-carrageenan, suggests that iota-carrageenan is desulfated enzymically to kappa-carrageenan.     

Comparison of chicken erythroid cell nuclear isolation methods using morphological,immunochemical and biochemical criteria

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.Abbreviations HMG high mobility group nonhistone chromosomal protein - RNP ribonucleoprotein - SSC 14 mM sodium citrate buffered saline pH 7.0 - PMSF phenylmethanesulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - DTT dithiothreitol - PBS 10 mM sodium phosphate buffered saline pH 7.2 - NP-40 Nonidet P-40 (octylphenoxypolyethoxyethanol) - SS-DNA single-stranded DNA - RSB reticulocyte standard buffer, 0.01 M NaCl, 0.003 M MgCl₂, 0.01 M Tris-HCI, pH 7.4.     

Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of Steel factor transgenic mice

Steel factor (SLF, also called KIT-ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro . In the present study, Kit-independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function-blocking antibody. We successfully purified these precursor or stem cells as Kit⁺CD45⁻ cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit-independent precursors. The Kit-independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit-independent nature for their survival.     

Membrane phospholipids, lipoxidative damage and molecular integrity: A causal role in aging and longevity

Nonenzymatic molecular modifications induced by reactive carbonyl species (RCS) generated by peroxidation of membrane phospholipids acyl chains play a causal role in the aging process. Most of the biological effects of RCS, mainly α,β-unsaturated aldehydes, di-aldehydes, and keto-aldehydes, are due to their capacity to react with cellular constituents, forming advanced lipoxidation end-products (ALEs). Compared to reactive oxygen and nitrogen species, lipid-derived RCS are stable and can diffuse within or even escape from the cell and attack targets far from the site of formation. Therefore, these soluble reactive intermediates, precursors of ALEs, are not only cytotoxic per se, but they also behave as mediators and propagators of oxidative stress and cellular and tissue damage. The consequent loss-of-function and structural integrity of modified biomolecules can have a wide range of downstream functional consequences and may be the cause of subsequent cellular dysfunctions and tissue damage. The causal role of ALEs in aging and longevity is inferred from the findings that follow: a) its accumulation with aging in several tissues and species; b) physiological interventions (dietary restriction) that increase longevity, decrease ALEs content; c) the longer the longevity of a species, the lower is the lipoxidation-derived molecular damage; and finally d) exacerbated levels of ALEs are associated with pathological states.     

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.     

Phosphate Immobilization by Oxide Precursors: Implications on Phosphate Availability before Life on Earth

The present study is based on the proposal that if the aqueous phosphorus-capture mechanism by iron oxide precursors was inhibited in prebiotic anoxic scenarios then soluble phosphates could have been more available than what is observed now. Supporting this conjecture, we examine prevailing contemporary trapping mechanisms of orthophosphate (Pi) and pyrophosphate (PPi). To illustrate its efficiency, the attachment of (Pi) ontoaggregates of iron-3 oxyhydroxide is compared with the one reported for the product of its condensation, PPi. The electrophoretic profiles of the Pi- and PPi-aggregatecomplexes reveal different pH-modulated interactions ofthe phosphorylated compounds with both the aggregate andits aqueous surrounding layers. The observed differencesof Pi/PPi sorption and desorption mechanisms are discussed in terms of their consequences to the prebiotic availability of soluble orthophosphate and of a phosphorylated compound having the high-energy phosphoanhydride linkage and a molecule representative of condensed oligophosphates.