Uptake of [3H]threonine in human colonic mucosa associated with carcinoma: An autoradiographic analysis at the ultrastructural level

Summary The uptake ofd,l-[G-³H]threonie was studied to show cellular protein synthesis, at the ultrastructural level, as part of our investigation into alterations in glycoprotein synthesis which occur in colonic mucosa adjacent to carcinoma (transitional mucosa). Threonine uptake, though variable, was higher in transitional than in normal mucosa. Most of the threonine was incorporated into the endoplasmic reticulum of the immature cells at the bottom of the crypt. With longer isotope incubation, activity was found in other organelles in cells which were still undifferentiated or immature or both.From our data, the increased uptake of [³H]threonine in transitional mucosa, seems to be the result of prolonged protein synthesis associated with an extension of the cellular proliferation zone in the crypt, rather than being the effect of increased cell turnover. Thus, variations in [³H]threonine uptake are not related to the changes in the rate of galactose incorporation in mucosa adjacent to carcinoma.     

Quantitative analysis of metabolite levels in normal human subjects by31P topical magnetic resonance

Conclusions The results we have obtained on forearm muscles of normal human subjects are complementary to those on isolated frog muscle and they lead, to the same conclusions. We are satisfied that our³¹P TMR measurements of [P-metabolites] in normal subjects are reliable, and we are now extending these methods for use in cases of degenerative muscular disorders (Edwards et al., 1982). Chemical estimates of [ADP] and [AMP] are not directly relevant to metabolic studies, and so an important contribution of NMR to the study of muscular mechanisms is its ability to estimate the concentrations of these metabolites that are free to take part in metabolic reactions.     

Cell kinetics of a chondrosarcoma.

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G1, 10-5 hr; S, 9-5 hr; G2, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0-5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.

Five glycosphingolipids (GSL), glucosylceramide, lactosylceramide, trihexosylceramide, globoside, and hematoside (GM3) were studied in serum from normal human subjects and patients with dyslipoproteinemia and found to be exclusively associated with the various classes of serum lipoproteins. Based on a unit weight of lipoprotein protein, the total amount of GSL in serum normal subjects was twice as high in very low density lipoprotein (VLDL) (d less than 1.006 g/ml) and low density lipoprotein (LDL) (d 1.019-1.063 g/ml) as in high density lipoproteins HDL2 (d 1.063-1.125 g/ml) or HDL3 (d 1.125-1.21 g/ml). In abetalipoproteinemia the levels of serum GSL were slightly reduced when compared to normal serum and were all found in the only existing lipoprotein, HDL; this contained 2-3 moles of GSL/ mole of lipoprotein as compared to 0.5 GSL/mole in normal HDL. In hypobetalipoproteinemia and Tangier disease, the serum glycosphingolipids were 10 to 30% reduced in concentration compared to the 75% reduction in other lipids, and were again found to be associated only with the serum lipoproteins. The relative proportions of GSL did not vary substantially in the normo- and hypolipidemic subjects studied. Only in patients with homozygous familial hypercholesterolemia was there a significant (3-4-fold) elevation of all of the five GSL species and this elevation of all of the five GSL species and this elevation correlated well with that of the circulating cholesterol and LDL. On a molar basis the LDL of these patients contained the same amount of GSL as normal subjects (5 moles GSL/mole protein). It is concluded that: (1) glycosphingolipids are associated only with the major lipoprotein classes in both normal and dyslipoproteinemic serum; (2) the relative proportions of the five glycosphingolipids are not significantly affected by dyslipoproteinemia; (3) only in severe hypolipoproteinemia do the remaining serum lipoproteins carry a complement of glycosphingolipids greater than normal. Although our results establish that glycosphingolipids are intimately associated with serum lipoproteins, the mode of association or the structural and functional significance of such an association remains undetermined.     

OLIGOSACCHARIDE STORAGE IN BRAINS FROM PATIENTS WITH FUCOSIDOSIS,GM1-GANGLIOSIDOSIS AND GM2-GANGLIOSIDOSIS (SANDHOFF'S DISEASE)1

Abstract— Analysis of whole autopsy brain from a patient with fucosidosis (α-fucosidase deficiency) revealed minor storage of H-antigen glycolipid [Fuc (α, 1→2) Gal-GlcNAc-Gal-Glc-Ceramide] and a slightly abnormal ganglioside composition in the form of a two-fold elevation of G_M1 and the presence of a fucose-containing glycolipid (a minor component) which co-migrated with G_D1a. The major storage materials in fucosidosis brain were an oligosaccharide (Fuc-Gal-GlcNAc-Man[Fuc-Gal-GlcNAc-Man]-ManGlcNAc) and a disaccharide [Fuc(α, 1→6)-GlcNAc] in the approximate ratio of 5:1. Lesser amounts of a related oligosaccharide (Gal-GlcNAc-Man[Gal-GlcNAc-Man]-Man-GlcNAc) were isolated from the brain of patients with G_M1-gangliosidosis (Types I and II) where the major storage material is known to be G_M1-ganglioside (Gal (β, 1→3)GalNAc(β, 1→4) [NeuNAcf(α, 2→3) Gal(β, 1→4)Glc-Ceramide). Similarly, a related oligosaccharide (GlcNAc-Man [GlcNAc-Man]-Man-GlcNAc) was isolated from the brain of a patient with a total deficiency of N-acetyl-β-d -hexosaminidase (Sandhoff variant of G_M2-gangliosidosis) where the major storage products are known to be G_M2-ganglioside (GalNAc (β 1→4) [NeuNAc (α, 2→3)Gal(β, 1→4)Glc-Ceramine) and its asialo derivative. These studies indicate that glycoproteins containing at least 2 mol of l -fucose per oligosaccharide unit are normally catabolized in human brain. Further, it appears that such glycoproteins are initially catabolized by an endo-N-acetylglucosaminidase to release an oligosaccharide which is then degraded by the sequential action of exo-glycosidases.     

Programmed cell death in neurotumour cells involves the generation of ceramide

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 µM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.Abbreviations PCD programmed cell death - PKC protein kinase C - HPTLC high-performance thin-layer chromatography - DETAPAC diethylenetriaminepentaacetic acid - DMEM Dubelco's modified Eagle's medium - FCS fetal calf serum - PBS phosphate-buffered saline - DAG diacylglycerol - DDI distilled-deionized - Cer ceramide - SM sphingomyelin Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Procedures for manufacturing double-barrelled ion-sensitive microelectrodes employing liquid sensors

Liquid ion-sensitive/selective sensors are available for most inorganic ions of physiological and biochemical importance. In order to measure intracellular ionic activities in relatively small cells, it is advisable to manufacture and use double-barrelled microelectrodes. Procedures for making two types of double-barrelled ion-sensitive microelectrode are described in detail. Such microelectrodes have been used successfully to measure intracellular K+, Cl- and Na+ activities in retinal horizontal cells of fish and body-wall muscles of insect larvae.     

Parkin‐independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease‐associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.