THE METABOLISM OF TISSUE CULTURES : I. PRELIMINARY STUDIES ON CHICK EMBRYO

1. The metabolism of chick embryo tissues has been followed by analysis of the culture media after various periods of incubation in roller bottles. 2. The initial rate of glucose utilization is increased by increasing glucose in the medium from 100 to 500 mg. per cent. Total glucose used can be increased in the same way or by daily addition of small amounts. Glucose is used in greatest amount when the medium containing 100 mg. per cent is replaced daily. 3. Although glucose consumption appears necessary for survival of cultures it may be used at a rate far in excess of that required for life and maximal growth. Complete blocking of mitosis by colchicine does not alter the rate of glucose utilization. 4. Proteolytic activity of the cultures is shown by an increase in the amino nitrogen of the peptone medium after incubation with tissue. 5. Utilization of nitrogen from an amino acid medium is shown by a decrease in the amino nitrogen of this medium. Cells obtaining their nitrogen from amino acids proliferate as rapidly as those grown in a medium identical except for the substitution of peptone, but the cell type is markedly different, in that embryo muscle forms cells resembling regenerating adult muscle. 6. Lactic acid was formed in both the presence and absence of glucose. Its formation increased with increased glucose utilization. There is some evidence that lactate may be utilized, and that it favors growth in the absence of glucose. 7. Added pyruvate was rapidly metabolized by the tissues. It, too, favors growth slightly in the absence of glucose.     

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester

Summary Ectoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis. In explants treated with phorbol 12-myristate 13-acetate the mitotic rate was increased.     

The genotoxicity of lucidin,a natural component of Rubia tinctorum L., and lucidinethylether,a component of ethanolic Rubia extracts

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HA hydroxyanthraquinones - LUE lucidinethylether - PRH primary rat hepatocytes - UDS unscheduled DNA synthesis     

Molecular cytogenetic characterization of two independent karyotypic anomalies in a patient with severe mental retardation and juvenile idiopathic arthritis

We report on a patient with severe mental retardation, dysmorphic features as well as juvenile idiopathic arthritis. G-banding indicated two independent karyotypic anomalies in this patient: an interstitial deletion del(X)(p21p22.3) and a rearrangement involving chromosomes 1 and 7, which represents a direct insertion, ins(7;1)(q36;p13.2p31.2). Non-random inactivation of the paternally derived del(X) chromosome was observed in blood lymphocytes and fibroblasts. High resolution analysis of the rearrangement involving chromosomes 1 and 7 subsequently revealed the additional submicroscopic deletion of at least 5 Mb at the 1p13.2 breakpoint. The deletion occurred on the paternal chromosome and encompasses the PTPN22 gene, already known to be associated with juvenile idiopathic arthritis. Our findings underline the importance of closely investigating the breakpoint regions of apparently balanced rearrangements in patients with abnormal phenotypes since complex chromosomal rearrangements (CCRs) may turn out to be unbalanced. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.     

Strategies for analysis of glycoprotein glycosylation

Glycoproteins are known to exhibit multiple biological functions. In order to assign distinct functional properties to defined structural features, detailed information on the respective carbohydrate moieties is required. Chemical and biochemical analyses, however, are often impeded by the small amounts of sample available and the vast structural heterogeneity of these glycans, thus necessitating highly sensitive and efficient methods for detection, separation and structural investigation. The aim of this article is to briefly review suitable strategies for characterization of glycosylation at the levels of intact proteins, glycopeptides and free oligosaccharides. Furthermore, methods commonly used for isolation, fractionation and carbohydrate structure analysis of liberated glycoprotein glycans are discussed in the context of potential applications in glycoproteomics.     

Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst

Zinc is an essential trace element for the immune system. It is known to be essential for highly proliferating cells, especially for cells of the immune system. However, zinc and other divalent cations are known to inhibit the human neutrophilic NADPH oxidase. Differentiated HL-60 cells were found to accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH oxidase by phorbol esters (PMA). This increase in vitamin C transport is due to the generation of superoxide and subsequent oxidation of ascorbate to dehydroascorbate (DHA) which is preferentially taken up by the cells. We found that zinc reversibly inhibits both PMA-stimulated ascorbate uptake and superoxide generation with a half-maximal effect at 20 microM of free zinc ions. Higher residual extracellular ascorbate concentrations were measured with increasing zinc concentrations, indicating that less ascorbate was oxidized and taken up by the cells. When the fluorescent dye diSC3(5) was used to monitor shifts in membrane potential, we found that depolarization with PMA was prolonged after preincubation of the cells with zinc. Suppression of the respiratory burst as well as inhibition of the uptake of the antioxidant vitamin C may disturb the balance between oxidative damage of invading particles and antioxidant protection in activated neutrophils.     

G to C transition at position −173 of MIF gene of the recipient is associated with reduced relapse rates after allogeneic stem cell transplantation

Pro-inflammatory and dendritic cell-activating properties of macrophage migration inhibitory factor (MIF) suggest a potentially important role for MIF in alloantigen-specific immune responses after allogeneic stem cell transplantation (allo-SCT). We tested whether MIF −173 G/C gene polymorphism of donor or patient had impacts on the outcomes after allo-SCT. Four hundred and fifty-four donor–patient pairs were genotyped and mortality, relapse, and development of complications were analyzed. Patient but not donor MIF −173*C allele was associated with improved overall survival (OS) (5 years: 60.8% versus 46.3%, p = 0.042) and disease free survival (DFS) (5 years: 55.4% versus 39.5%; p = 0.014) due to a reduction in relapse (day 2000: 22.8% versus 42.0% p = 0.006) but not due to decreased transplantation-related mortality (TRM) (p = 0.44). Multivariate analysis proved patient −173*C allele as an independent factor for reducing relapse after allo-SCT (p = 0.023). Subgroup analysis showed a clear MIF −173*C allele-related reduction in relapse for those patients who did not receive T cell depleted (TCD) SCT (p = 0.01) in contrast to patients receiving TCD SCT (p = 0.20). In summary, patient MIF −173*C allele may be linked to specific, yet unrevealed functions in tumor biology and graft versus leukemia and lymphoma effects and potentially presents a novel prognostic marker for patient-tailored counseling and therapy in allo-SCT.